Aminopyridine compounds and methods for the preparation and use thereof

ABSTRACT

The present invention relates generally to therapeutics targeting the bacterium Porphyromonas gingivalis, including its proteases arginine gingipain A/B (Rgp), and their use for the treatment of disorders associated with P. gingivalis infection, including brain disorders such as Alzheimer&#39;s disease. In certain embodiments, the invention provides compounds according to Formula I and Formula III, as described herein, and pharmaceutically acceptable salts thereof.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/678,700, filed on Nov. 8, 2019, which is a continuation ofInternational Patent Application No. PCT/US2018/032139, filed on May 10,2018, which claims priority to U.S. Provisional Pat. Appl. No.62/504,442, filed on May 10, 2017, and U.S. Provisional Pat. Appl. No.62/504,480, filed on May 10, 2017, which applications are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

Infection with the bacterium Porphyromonas gingivalis has been linked tothe development of periodontal disease, Alzheimer's disease and otherbrain disorders, cardiovascular disease, diabetes, cancer, liverdisease, kidney disease, preterm birth, arthritis, pneumonia and otherdisorders. P. gingivalis is an anaerobic asaccharolytic gram-negativerod bacterium that is known to infect the oral cavity and translocatesystemically into coronary arteries, aorta, placental tissue, the brain,the kidneys, and the liver. The bacterium has also been identified incancerous tissues and a mechanism has been proposed by which gingipainscan trigger immortalization and metastasis. See: Gandhimadhi, et al.Journal of Indian Society of Periodontology. 2010; 14(2): 114-120; Liao,et al, Med Hypotheses, 2009. 72(6): 732-5; Byrne, et al., Oral MicrobiolImmunol, 2009. 24(6): 469-77; Mahendra, et al., J Maxillofac Oral Surg,2009. 8(2): 108-13; Stelzel, et al., J Periodontol, 2002. 73(8): 868-70;Katz, et al., Journal of Dental Research, 2009. 88(6): 575-578; Poole,et al., J Alzheimers Dis, 2015, 43(1): 67-80; Ishikawa, et al., BiochimBiophys Acta, 2013. 1832(12): 2035-2043; Inaba, et al., CellularMicrobiology, 2014. 16(1): 131-145.

P. gingivalis produces proteases called gingipains, including ArginineGingipain A (RgpA), Arginine Gingipain B (RgpB) and Lysine Gingipain(Kgp). Gingipains contribute to many functions of the organism includingits survival and virulence. Gingipains can be secreted, transported toouter membrane surfaces of P. gingivalis, or released in outer membranevesicles by the bacterium. Gingipains degrade a broad range of proteins(e.g., immunoglobulins, proteinase inhibitors, actin, and collagen)which can lead to cytoskeleton collapse and apoptosis in many types ofcells. Recent research has demonstrated that inhibitors of gingipainscan prevent P. gingivalis-induced cell death. See; Travis, et al., AdvExp Med Biol, 2000. 477: 455-65; Sheets, et al., Infect Immun, 2005.73(3): 1543-52; Sheets, et al., Infect Immun, 2006. 74(10): 5667-78;Stathopoulou, et al., BMC Microbiol, 2009. 9: 107. New compounds for theinhibition of gingipain activity and the treatment of diseasesassociated with gingipain activity and P. gingivalis infection areneeded. The present invention addresses this and other needs.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the invention provides a compound according toFormula I:

-   -   or a pharmaceutically acceptable salt thereof, wherein    -   W is selected from the group consisting of a bond, CH₂, and O;    -   R^(1a) and R^(1b) are independently selected from the group        consisting of H and C₁₋₆ alkyl;    -   R² is selected from the group consisting of C₁₋₆ alkyl and        halogen;    -   subscript n is 0 or 1;    -   R³ is selected from the group consisting of C₃₋₈ alkyl, C₃₋₈        cycloalkyl, C₃₋₁₂ heterocyclyl, C₆₋₁₀ aryl, and C₅₋₁₂ heteroaryl        wherein R³ is optionally substituted with one or more R^(3a)        substituents;    -   each R^(3a) is independently selected from the group consisting        of        -   halogen, —CN, —NO₂, —N₃, —OH, C₁₋₄ alkyl, C₁₋₄ haloalkyl,            C₁₋₄ alkoxy, C₁₋₄ haloalkoxy, —N(R^(c))₂,            —(CH₂)_(k)C(O)R^(b), —NR^(c)(CH₂)_(u)C(O)R^(b),            —O(CH₂)_(u)C(O)R^(b), —(CH₂)_(k)CONR^(c)R^(c),            —(CH₂)_(k)NR^(c)C(O)R^(b), —NR^(c)(CH₂)_(u)CONR^(c)R^(c),            —NR^(c)(CH₂)_(u)NR^(c)C—(O)R^(b), —O(CH₂)_(u)CONR^(c)R^(c),        -   and —O(CH₂)_(u)NR^(c)C(O)R^(b), and optionally substituted            triazolyl;    -   each R^(b) is independently selected from the group consisting        of C₁₋₄ alkyl, C₁₋₄ haloalkyl, and C₁₋₄ deuteroalkyl;    -   each R^(c) is independently selected from the group consisting        of hydrogen and C₁₋₈ alkyl;    -   each subscript k is independently selected from 0, 1, 2, 3, 4,        5, and 6;    -   each subscript u is independently selected from 1, 2, 3, 4, 5,        and 6;    -   R⁴ is selected from the group consisting of —CH₂R^(4a) and C₁₋₆        haloalkyl;    -   R^(4a) is selected from the group consisting of —O—R⁵, —S—R⁶,        —SO—R⁶, —SO₂—R⁶, —N(R⁷)₂, and C₅₋₁₂ heteroaryl;    -   R⁵ is selected from the group consisting of phenyl, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, and C₅₋₁₂ heteroaryl, wherein phenyl is        substituted with 1-5 halogens, and wherein C₅₋₁₂ heteroaryl is        optionally substituted with halogen or C₁₋₃ haloalkyl;    -   R⁶ is selected from the group consisting of phenyl, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, and C₅₋₁₂ heteroaryl, wherein phenyl is        optionally substituted with 1-5 halogens, and wherein C₅₋₁₂        heteroaryl is optionally substituted with halogen or C₁₋₃        haloalkyl; and    -   each R⁷ is independently selected C₁₋₄ alkyl.

In one embodiment, the invention provides a compound according toFormula III:

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹¹ is selected from C₁₋₆ alkyl and C₃₋₈ cycloalkyl;    -   R^(12a) and R^(12b) are independently selected from H, C₁₋₆        alkyl, and C₆₋₁₀ aryl, or        -   R^(12a) and R^(12b) are taken together to form C₃₋₆            cycloalkyl, or        -   R^(12a) and R¹¹ are taken together to form 4- to 10-membered            heterocyclyl which is optionally substituted with one or            more R¹⁷;    -   each R^(13a) and each R^(13b) is independently selected from H,        —OH, and C₁₋₆ alkyl, or        -   one R^(13a) and R¹¹ are taken together to form 4- to            10-membered heterocyclyl, or        -   one R^(13b) and R^(12b) are taken together to form a 5- or            6-membered ring;    -   R¹⁴ is selected from H and halogen, or        -   R¹⁴, R^(12a), and R^(12b) are taken together to form a 6- to            8-membered ring, which is optionally substituted with one or            more R¹⁸, or        -   R¹⁴ and one R^(13a) are taken together to form a 5- to            8-membered ring, which is optionally substituted with one or            more R¹⁸, or        -   R¹⁴ is taken together with one R^(13a) and one R^(13b) on            the same carbon atom to form a 5- to 8-membered ring, which            is optionally substituted with one or more R¹⁸, or        -   R¹⁴, R¹¹, and R^(12a) are taken together to form a 6- to            10-membered bicyclic ring, which is optionally substituted            with one or more R¹⁸;    -   R^(15a) and R^(15b) are independently selected from H and C₁₋₆        alkyl;    -   R¹⁶ is independently selected from C₁₋₆ alkyl and halogen;    -   each R¹⁷ is independently selected from C₁₋₆ alkyl, C₁₋₆ alkoxy,        C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, —OH, and —N(R^(17a))₂, wherein        each R^(17a) is independently selected from H and C₁₋₆ alkyl;    -   each R¹⁸ is independently selected from C₁₋₆ alkyl and halogen;    -   Y is selected from O, S, C(R^(19a))₂, and NR^(19b);    -   each R^(19a) is selected from H and C₁₋₆ alkyl, or        -   one R^(19a) and one R^(13b) on adjacent atoms are taken            together to form a double bond; R^(19b) is selected from H            and C₁₋₆ alkyl, or        -   R^(19b) and R¹¹ are taken together to form a 4- to            6-membered ring;    -   subscript m is 0, 1, 2, or 3; and    -   subscript q is 0 or 1.

In a related embodiment, the invention provides a pharmaceuticalcomposition containing a compound as described herein, or apharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable excipients.

In another embodiment, the invention provides a method of inhibiting agingipain. The method includes contacting the gingipain with aneffective amount of a compound as described herein.

In another embodiment, the invention provides a method of treating adisease or condition associated with P. gingivalis infection. The methodincludes administering to a subject in need thereof an effective amountof a compound or pharmaceutical composition as described herein.

DETAILED DESCRIPTION OF THE INVENTION I. General

Inhibition of gingipains has been shown to protect cells, preventbacterial growth, increase immune system surveillance of P. gingivalis,and protect against bacterial reinfection. The present inventionprovides potent nonpeptidic compounds for inhibition of argininegingipains. The compounds can be used to prevent cell death,inflammation, and other pathology in a variety of diseases associatedwith P. gingivalis infection, including aging-related conditions such asAlzheimer's disease.

II. Definitions

As used herein, the term “alkyl,” by itself or as part of anothersubstituent, refers to a straight or branched, saturated, aliphaticradical having the number of carbon atoms indicated. Alkyl can includeany number of carbons, such as C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈,C₁₋₉, C₁₋₁₀, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ andC₅₋₆. For example, C₁₋₆ alkyl includes, but is not limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groupshaving up to 20 carbons atoms, such as, but not limited to heptyl,octyl, nonyl, decyl, etc. Alkyl groups can be substituted orunsubstituted. For example, “substituted alkyl” groups can besubstituted with one or more groups selected from halo, hydroxy, amino,alkylamino, amido, acyl, nitro, cyano, and alkoxy.

As used herein, the term “alkoxy,” by itself or as part of anothersubstituent, refers to a group having the formula —OR, wherein R isalkyl.

As used herein, the term “cycloalkyl,” by itself or as part of anothersubstituent, refers to a saturated or partially unsaturated, monocyclic,fused bicyclic or bridged polycyclic ring assembly containing from 3 to12 ring atoms, or the number of atoms indicated. Cycloalkyl can includeany number of carbons, such as C₃₋₆, C₄₋₆, C₅₋₆, C₃₋₈, C₄₋₈, C₅₋₈, C₆₋₈,C₃₋₉, C₃₋₁₀, C₃₋₁₁, and C₃₋₁₂. Saturated monocyclic cycloalkyl ringsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl ringsinclude, for example, norbornane, [2.2.2] bicyclooctane,decahydronaphthalene and adamantane. Cycloalkyl groups can also bepartially unsaturated, having one or more double or triple bonds in thering. Representative cycloalkyl groups that are partially unsaturatedinclude, but are not limited to, cyclobutene, cyclopentene, cyclohexene,cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene,cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene,and norbornadiene. When cycloalkyl is a saturated monocyclic C₃₋₈cycloalkyl, exemplary groups include, but are not limited tocyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl andcyclooctyl. When cycloalkyl is a saturated monocyclic C₃₋₆ cycloalkyl,exemplary groups include, but are not limited to cyclopropyl,cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can besubstituted or unsubstituted. For example, “substituted cycloalkyl”groups can be substituted with one or more groups selected from halo,hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

As used herein, the term “alkylene” refers to an alkyl group, as definedabove, linking at least two other groups (i.e., a divalent alkylradical). The two moieties linked to the alkylene group can be linked tothe same carbon atom or different carbon atoms of the alkylene group.

As used herein, the term “alkylthio,” by itself or as part of anothersubstituent, refers to a group having the formula —SR, wherein R isalkyl.

As used herein, the term “heteroalkyl,” by itself or as part of anothersubstituent, refers to an alkyl group of any suitable length and havingfrom 1 to 3 heteroatoms such as N, O and S. For example, heteroalkyl caninclude ethers, thioethers and alkyl-amines. Additional heteroatoms canalso be useful, including, but not limited to, B, Al, Si and P. Theheteroatoms can be oxidized to form moieties such as, but not limitedto, —S(O)— and —S(O)₂—. The heteroatom portion of the heteroalkyl canreplace a hydrogen of the alkyl group to form a hydroxy, thio, or aminogroup. Alternatively, the heteroatom portion can be the connecting atom,or be inserted between two carbon atoms.

As used herein, the terms “halo” and “halogen,” by themselves or as partof another substituent, refer to a fluorine, chlorine, bromine, oriodine atom.

As used herein, the term “haloalkyl,” by itself or as part of anothersubstituent, refers to an alkyl group where some or all of the hydrogenatoms are replaced with halogen atoms. As for alkyl groups, haloalkylgroups can have any suitable number of carbon atoms, such as C₁₋₆. Forexample, haloalkyl includes trifluoromethyl, fluoromethyl, etc. In someinstances, the term “perfluoro” can be used to define a compound orradical where all the hydrogens are replaced with fluorine. For example,perfluoromethyl refers to 1,1,1-trifluoromethyl.

As used herein, the term “haloalkoxy,” by itself or as part of anothersubstituent, refers to an alkoxy group where some or all of the hydrogenatoms are replaced with halogen atoms.

As used herein, the term “halocycloalkyl,” by itself or as part ofanother substituent, refers to a cycloalkyl group where some or all ofthe hydrogen atoms are replaced with halogen atoms.

As used herein, the term “deuteroalkyl,” by itself or as part of anothersubstituent, refers to an alkyl group where some or all of the hydrogenatoms are replaced with deuterium atoms. As for alkyl groups,deuteroalkyl groups can have any suitable number of carbon atoms, suchas C₁₋₆. In some instances, the term “perdeutero” can be used to definea compound or radical where all the hydrogens are replaced withdeuterium.

As used herein, the term “aryl,” by itself or as part of anothersubstituent, refers to an aromatic ring system having any suitablenumber of carbon ring atoms and any suitable number of rings. Arylgroups can include any suitable number of carbon ring atoms, such as C₆,C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅ or C₁₆, as well as C₆₋₁₀,C₆₋₁₂, or C₆₋₁₄. Aryl groups can be monocyclic, fused to form bicyclic(e.g., benzocyclohexyl) or tricyclic groups, or linked by a bond to forma biaryl group. Representative aryl groups include phenyl, naphthyl andbiphenyl. Other aryl groups include benzyl, having a methylene linkinggroup. Some aryl groups have from 6 to 12 ring members, such as phenyl,naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members,such as phenyl or naphthyl. Some other aryl groups have 6 ring members,such as phenyl. Aryl groups can be substituted or unsubstituted. Forexample, “substituted aryl” groups can be substituted with one or moregroups selected from halo, hydroxy, amino, alkylamino, amido, acyl,nitro, cyano, and alkoxy.

As used herein, the term “heteroaryl,” by itself or as part of anothersubstituent, refers to a monocyclic or fused bicyclic or tricyclicaromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5of the ring atoms are a heteroatom such as N, O or S. Additionalheteroatoms can also be useful, including, but not limited to, B, Al, Siand P. The heteroatoms can be oxidized to form moieties such as, but notlimited to, —S(O)— and —S(O)₂—. Heteroaryl groups can include any numberof ring atoms, such as C₅₋₆, C₃₋₈, C₄₋₈, C₅₋₈, C₆₋₈, C₃₋₉, C₃₋₁₀, C₃₋₁₁,or C₃₋₁₂, wherein at least one of the carbon atoms is replaced by aheteroatom. Any suitable number of heteroatoms can be included in theheteroaryl groups, such as 1, 2, 3, 4; or 5, or 1 to 2, 1 to 3, 1 to 4,1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, or 3 to 5. For example,heteroaryl groups can be C₅₋₈ heteroaryl, wherein 1 to 4 carbon ringatoms are replaced with heteroatoms; or C₅₋₈ heteroaryl, wherein 1 to 3carbon ring atoms are replaced with heteroatoms; or C₅₋₆ heteroaryl,wherein 1 to 4 carbon ring atoms are replaced with heteroatoms; or C₅₋₆heteroaryl, wherein 1 to 3 carbon ring atoms are replaced withheteroatoms. The heteroaryl group can include groups such as pyrrole,pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine,pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers),thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. Theheteroaryl groups can also be fused to aromatic ring systems, such as aphenyl ring, to form members including, but not limited to,benzopyrroles such as indole and isoindole, benzopyridines such asquinoline and isoquinoline, benzopyrazine (quinoxaline), benzopyrimidine(quinazoline), benzopyridazines such as phthalazine and cinnoline,benzothiophene, and benzofuran. Other heteroaryl groups includeheteroaryl rings linked by a bond, such as bipyridine. Heteroaryl groupscan be substituted or unsubstituted. For example, “substitutedheteroaryl” groups can be substituted with one or more groups selectedfrom halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, andalkoxy.

The heteroaryl groups can be linked via any position on the ring. Forexample, pyrrole includes 1-, 2- and 3-pyrrole, pyridine includes 2-, 3-and 4-pyridine, imidazole includes 1-, 2-, 4- and 5-imidazole, pyrazoleincludes 1-, 3-, 4- and 5-pyrazole, triazole includes 1-, 4- and5-triazole, tetrazole includes 1- and 5-tetrazole, pyrimidine includes2-, 4-, 5- and 6-pyrimidine, pyridazine includes 3- and 4-pyridazine,1,2,3-triazine includes 4- and 5-triazine, 1,2,4-triazine includes 3-,5- and 6-triazine, 1,3,5-triazine includes 2-triazine, thiopheneincludes 2- and 3-thiophene, furan includes 2- and 3-furan, thiazoleincludes 2-, 4- and 5-thiazole, isothiazole includes 3-, 4- and5-isothiazole, oxazole includes 2-, 4- and 5-oxazole, isoxazole includes3-, 4- and 5-isoxazole, indole includes 1-, 2- and 3-indole, isoindoleincludes 1- and 2-isoindole, quinoline includes 2-, 3- and 4-quinoline,isoquinoline includes 1-, 3- and 4-isoquinoline, quinazoline includes 2-and 4-quinoazoline, cinnoline includes 3- and 4-cinnoline,benzothiophene includes 2- and 3-benzothiophene, and benzofuran includes2- and 3-benzofuran.

Some heteroaryl groups include those having from 5 to 10 ring membersand from 1 to 3 ring atoms including N, O or S, such as pyrrole,pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine,pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene,furan, thiazole, isothiazole, oxazole, isoxazole, indole, isoindole,quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine,cinnoline, benzothiophene, and benzofuran. Other heteroaryl groupsinclude those having from 5 to 8 ring members and from 1 to 3heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole,pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, andisoxazole. Some other heteroaryl groups include those having from 9 to12 ring members and from 1 to 3 heteroatoms, such as indole, isoindole,quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine,cinnoline, benzothiophene, benzofuran and bipyridine. Still otherheteroaryl groups include those having from 5 to 6 ring members and from1 to 2 ring atoms including N, O or S, such as pyrrole, pyridine,imidazole, pyrazole, pyrazine, pyrimidine, pyridazine, thiophene, furan,thiazole, isothiazole, oxazole, and isoxazole.

Some heteroaryl groups include from 5 to 10 ring members and onlynitrogen heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole,triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), indole, isoindole, quinoline, isoquinoline, quinoxaline,quinazoline, phthalazine, and cinnoline. Other heteroaryl groups includefrom 5 to ring members and only oxygen heteroatoms, such as furan andbenzofuran. Some other heteroaryl groups include from 5 to 10 ringmembers and only sulfur heteroatoms, such as thiophene andbenzothiophene. Still other heteroaryl groups include from 5 to 10 ringmembers and at least two heteroatoms, such as imidazole, pyrazole,triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), thiazole, isothiazole, oxazole, isoxazole, quinoxaline,quinazoline, phthalazine, and cinnoline.

As used herein, the term “heterocyclyl,” by itself or as part of anothersubstituent, refers to a saturated ring system having from 3 to 12 ringmembers and from 1 to 4 heteroatoms of N, O and S. Additionalheteroatoms can also be useful, including, but not limited to, B, Al, Siand P. The heteroatoms can be oxidized to form moieties such as, but notlimited to, —S(O)— and —S(O)₂—. Heterocyclyl groups can include anynumber of ring atoms, such as, C₃₋₆, C₄₋₆, C₅₋₆, C₃₋₈, C₄₋₈, C₅₋₈, C₆₋₈,C₃₋₉, C₃₋₁₀, C₃₋₁₁, or C₃₋₁₂, wherein at least one of the carbon atomsis replaced by a heteroatom. Any suitable number of carbon ring atomscan be replaced with heteroatoms in the heterocyclyl groups, such as 1,2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. Theheterocyclyl group can include groups such as aziridine, azetidine,pyrrolidine, piperidine, azepane, azocane, quinuclidine, pyrazolidine,imidazolidine, piperazine (1,2-, 1,3- and 1,4-isomers), oxirane,oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane,thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran),oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane,dithiolane, morpholine, thiomorpholine, dioxane, or dithiane. Theheterocyclyl groups can also be fused to aromatic or non-aromatic ringsystems to form members including, but not limited to, indoline.Heterocyclyl groups can be unsubstituted or substituted. For example,“substituted heterocyclyl” groups can be substituted with one or moregroups selected from halo, hydroxy, amino, oxo, alkylamino, amido, acyl,nitro, cyano, and alkoxy.

The heterocyclyl groups can be linked via any position on the ring. Forexample, aziridine can be 1- or 2-aziridine, azetidine can be 1- or2-azetidine, pyrrolidine can be 1-, 2- or 3-pyrrolidine, piperidine canbe 1-, 2-, 3- or 4-piperidine, pyrazolidine can be 1-, 2-, 3-, or4-pyrazolidine, imidazolidine can be 1-, 2-, 3- or 4-imidazolidine,piperazine can be 1-, 2-, 3- or 4-piperazine, tetrahydrofuran can be 1-or 2-tetrahydrofuran, oxazolidine can be 2-, 3-, 4- or 5-oxazolidine,isoxazolidine can be 2-, 3-, 4- or 5-isoxazolidine, thiazolidine can be2-, 3-, 4- or 5-thiazolidine, isothiazolidine can be 2-, 3-, 4- or5-isothiazolidine, and morpholine can be 2-, 3- or 4-morpholine.

When heterocyclyl includes 3 to 8 ring members and 1 to 3 heteroatoms,representative members include, but are not limited to, pyrrolidine,piperidine, tetrahydrofuran, oxane, tetrahydrothiophene, thiane,pyrazolidine, imidazolidine, piperazine, oxazolidine, isoxazolidine,thiazolidine, isothiazolidine, morpholine, thiomorpholine, dioxane anddithiane. Heterocyclyl can also form a ring having 5 to 6 ring membersand 1 to 2 heteroatoms, with representative members including, but notlimited to, pyrrolidine, piperidine, tetrahydrofuran,tetrahydrothiophene, pyrazolidine, imidazolidine, piperazine,oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, andmorpholine.

As used herein, the term “protecting group” refers to a chemical moietythat renders a functional group (e.g., an amino group) unreactive, butis also removable so as to restore the amino group. Examples ofprotecting groups include, but are not limited to, benzyloxycarbonyl (Zor Cbz); 9-fluorenylmethyloxycarbonyl (Fmoc); tert-butyloxycarbonyl(Boc); allyloxycarbonyl (Alloc); p-toluene sulfonyl (Tos);2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc);2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf);mesityl-2-sulfonyl (Mts); 4-methoxy-2,3,6-trimethylphenylsulfonyl (Mtr);acetamido; phthalimido; and the like. Other protecting groups are knownto those of skill in the art including, for example, those described byGreen and Wuts (Protective Groups in Organic Synthesis, 4^(th) Ed 2007,Wiley-Interscience, New York).

As used herein, the term “carbonyl,” by itself or as part of anothersubstituent, refers to —C(O)—, i.e., a carbon atom double-bonded tooxygen and bound to two other groups in the moiety having the carbonyl.

As used herein, the term “amino” refers to a moiety —NR₂, wherein each Rgroup is H or alkyl. An amino moiety can be ionized to form thecorresponding ammonium cation. “Dialkylamino” refers to an amino moietywherein each R group is alkyl.

As used herein, the term “sulfonyl” refers to a moiety —SO₂R, whereinthe R group is alkyl, haloalkyl, or aryl. An amino moiety can be ionizedto form the corresponding ammonium cation. “Alkylsulfonyl” refers to anamino moiety wherein the R group is alkyl.

As used herein, the term “hydroxy” refers to the moiety —OH.

As used herein, the term “cyano” refers to a carbon atom triple-bondedto a nitrogen atom (i.e., the moiety —C≡N).

As used herein, the term “carboxy” refers to the moiety —C(O)OH. Acarboxy moiety can be ionized to form the corresponding carboxylateanion.

As used herein, the term “amido” refers to a moiety —NRC(O)R or—C(O)NR₂, wherein each R group is H or alkyl.

As used herein, the term “nitro” refers to the moiety —NO₂.

As used herein, the term “oxo” refers to an oxygen atom that isdouble-bonded to a compound (i.e., O═).

In general, the term “substituted,” whether preceded by the term“optionally” or not, means that one or more hydrogens of the designatedmoiety are replaced with a suitable substituent. Unless otherwiseindicated, an “optionally substituted” group may have a suitablesubstituent at each substitutable position of the group, and when morethan one position in any given structure may be substituted with morethan one substituent selected from a specified group, the substituentmay be either the same or different at every position. Combinations ofsubstituents are generally those that result in the formation of stableor chemically feasible compounds. The term “stable,” as used herein,refers to compounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein. In general, “substituted,” as usedherein, does not encompass replacement and/or alteration of a keyfunctional group by which a molecule is identified, e.g., such that the“substituted” functional group becomes, through substitution, adifferent functional group. For example, a “substituted phenyl” groupmust still comprise the phenyl moiety and cannot be modified bysubstitution, in this definition, to become, e.g., a cyclohexyl group.

Examples of suitable monovalent substituents on a substitutable carbonatom of an “optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(α); —(CH₂)₀₋₄OR^(α); —O(CH₂)₀₋₄R^(α), —O—(CH₂)₀₋₄C(O)OR^(α);—(CH₂)₀₋₄CH(OR^(α))₂; —(CH₂)₀₋₄SR^(α); —(CH₂)₀₋₄Ph, wherein Ph is phenylwhich may be substituted with R^(α); —(CH₂)₀₋₄O(CH₂)₀₋₁phenyl, whichphenyl may be substituted with R^(α); —CH═CHPh, wherein Ph is phenylwhich may be substituted with R^(α); —(CH₂)₀₋₄O(CH₂)₀₋₁-Py, wherein Pyis pyridyl which may be substituted with R^(α); —NO₂; —CN; —N₃;—(CH₂)₀₋₄N(R^(α))₂; —(CH₂)₀₋₄N(R^(α))C(O)R^(α); —N(R^(α))C(S)R^(α);—(CH₂)₀₋₄N(R^(α))C(O)NR^(α) ₂; —N(R^(α))C(S)NR^(α) ₂;—(CH₂)₀₋₄N(R^(α))C(O)OR^(α); —N(R^(α))N(R^(α))C(O)R^(α);—N(R^(α))N(R^(α))C(O)NR^(α) ₂; —N(R^(α))N(R^(α))C(O)OR^(α);—(CH₂)₀₋₄C(O)R^(α); —C(S)R^(α); —(CH₂)₀₋₄C(O)OR^(α);—(CH₂)₀₋₄C(O)SR^(α); —(CH₂)₀₋₄C(O)OSiR^(α) ₃; —(CH₂)₀₋₄OC(O)R^(α);—OC(O)(CH₂)₀₋₄SR—SC(S)SR^(α); —(CH₂)₀₋₄SC(O)R^(α); —(CH₂)₀₋₄C(O)NR^(α)₂; —C(S)NR^(α) ₂, —C(S)SR^(α); —SC(S)SR^(α), —(CH₂)₀₋₄OC(O)NR^(α) ₂;—C(O)N(OR^(α))R^(α); —C(O)C(O)R^(α); —C(O)CH₂C(O)R^(α);—C(NOR^(α))R^(α); —(CH₂)₀₋₄SSR^(α); —(CH₂)₀₋₄S(O)₂R^(α);—(CH₂)₀₋₄S(O)₂OR^(α); —(CH₂)₀₋₄OS(O)₂R^(α); —S(O)₂NR^(α) ₂;—(CH₂)₀₋₄S(O)R^(α); —N(R^(α))S(O)₂NR^(α) ₂; —N(R^(α))S(O)₂R^(α);—N(OR^(α))R^(α); —C(NH)NR^(α) ₂; —P(O)₂R^(α); —P(O)R^(α) ₂; —OP(O)R^(a)₂; —OP(O)(OR^(α))₂; SiR^(α) ₃; —(C₁₋₄ straight orbranched)alkylene)-O—N(R^(α))₂; or —(C₁₋₄ straight orbranched)alkylene)C(O)O—N(R^(α))₂. Each R^(α) is independently hydrogen;C₁₋₆ alkyl; —CH₂Ph, —O(CH₂)₀₋₁Ph; —CH₂-(5- to 6-membered heteroaryl);C₃₋₈ cycloalkyl; C₆₋₁₀ aryl; 4- to 10-membered heterocyclyl; or 6- to10-membered heteroaryl; and each R^(α) may be further substituted asdescribed below.

Examples of suitable monovalent substituents on R^(α) are independentlyhalogen, —(CH₂)₀₋₂R^(β); —(CH₂)₀₋₂OH; —(CH₂)₀₋₂OR^(β);—(CH₂)₀₋₂CH(OR^(β))₂; —CN; —N₃; —(CH₂)₀₋₂C(O)R^(β); —(CH₂)₀₋₂C(O)OH;—(CH₂)₀₋₂C(O)OR^(β); —(CH₂)₀₋₂SR^(β); —(CH₂)₀₋₂SH; —(CH₂)₀₋₂NH₂;—(CH₂)₀₋₂NHR^(β); —(CH₂)₀₋₂NR^(β) ₂; —NO₂; SiR^(β) ₃; —OSiR^(β) ₃;—C(O)SR^(β); —(C₁₋₄ straight or branched alkylene)C(O)OR^(β); or—SSR^(β); wherein each R^(β) is independently selected from C₁₋₄ alkyl;—CH₂Ph; —O(CH₂)₀₋₁Ph; C₃₋₈ cycloalkyl; C₆₋₁₀ aryl; 4- to 10-memberedheterocyclyl; or 6- to 10-membered heteroaryl. Suitable divalentsubstituents on a saturated carbon atom of R^(α) include ═O and ═S.

Examples of suitable divalent substituents on a saturated carbon atom ofan “optionally substituted” group include the following: ═O; ═S;═NNR^(γ) ₂; ═NNHC(O)R^(γ); ═NNHC(O)OR^(γ); ═NNHS(O)₂R^(γ); ═NR^(γ);═NOR^(γ); —O(C(R^(γ) ₂))₂₋₃O—; or —S(C(R^(γ) ₂))₂₋₃S—; wherein eachindependent occurrence of R^(γ) is selected from hydrogen; C₁₋₆ alkyl,which may be substituted as defined below; C₃₋₈ cycloalkyl; C₆₋₁₀ aryl;4- to 10-membered heterocyclyl; or 6- to 10-membered heteroaryl.Suitable divalent substituents that are bound to vicinal substitutablecarbons of an “optionally substituted” group include: —O(CR^(β) ₂)₂₋₃O—;wherein each independent occurrence of R^(β) is selected from hydrogen;C₁₋₆ alkyl which may be substituted as defined below; C₃₋₈ cycloalkyl;C₆₋₁₀ aryl; 4- to 10-membered heterocyclyl; or 6- to 10-memberedheteroaryl.

Examples of suitable substituents on the alkyl group of R^(γ) includehalogen; —R^(δ); —OH; —OR^(δ); —CN; —C(O)OH; —C(O)OR^(δ); —NH₂;—NHR^(δ); —NR^(δ) ₂; or —NO₂; wherein each R^(δ) is independently C₁₋₄alkyl; —CH₂Ph; —O(CH₂)₀₋₁Ph; 4- to 10-membered heterocyclyl; or 6- to10-membered heteroaryl.

Examples of suitable substituents on a substitutable nitrogen of an“optionally substituted” group include —R^(ε); —NR^(ε) ₂; —C(O)R^(ε);—C(O)OR^(ε); —C(O)C(O)R^(ε); —C(O)CH₂C(O)R^(ε); —S(O)₂R^(ε);—S(O)₂NR^(ε) ₂; —C(S)NR^(ε) ₂; —C(NH)NR^(ε) ₂; or —N(R^(ε))S(O)₂R^(ε);wherein each R^(δ) is independently hydrogen; C₁₋₆ alkyl which may besubstituted as defined below; C₃₋₈ cycloalkyl; C₆₋₁₀ aryl; 4- to10-membered heterocyclyl; or 6- to 10-membered heteroaryl.

Examples of suitable substituents on the alkyl group of R^(ε) areindependently halogen; —R^(δ); —OH; —OR^(δ); —CN; —C(O)OH; —C(O)OR^(δ);—NH₂; —NHR^(δ); —NR^(δ) ₂; or —NO₂; wherein each R^(δ) is independentlyC₁₋₄ alkyl; —CH₂Ph; —O(CH₂)₀₋₁Ph; C₆₋₁₀ aryl; 4- to 10-memberedheterocyclyl; or 6- to 10-membered heteroaryl.

As used herein, the term “pharmaceutically acceptable excipient” refersto a substance that aids the administration of an active agent to asubject. By “pharmaceutically acceptable,” it is meant that theexcipient is compatible with the other ingredients of the formulationand is not deleterious to the recipient thereof. Pharmaceuticalexcipients useful in the present invention include, but are not limitedto, binders, fillers, disintegrants, lubricants, glidants, coatings,sweeteners, flavors and colors.

As used herein, the term “salt” refers to acid or base salts of thecompounds of the invention. Illustrative examples of pharmaceuticallyacceptable salts are mineral acid (hydrochloric acid, hydrobromic acid,phosphoric acid, and the like) salts, organic acid (acetic acid,propionic acid, glutamic acid, citric acid and the like) salts, andquaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.It is understood that the pharmaceutically acceptable salts arenon-toxic.

Pharmaceutically acceptable salts of the acidic compounds of the presentinvention are salts formed with bases, namely cationic salts such asalkali and alkaline earth metal salts (such as sodium, lithium,potassium, calcium, and magnesium salts), as well as ammonium salts(such as ammonium, trimethyl-ammonium, diethylammonium, andtris-(hydroxymethyl)-methyl-ammonium salts).

Similarly acid addition salts, such as of mineral acids, organiccarboxylic and organic sulfonic acids, e.g., hydrochloric acid,methanesulfonic acid, maleic acid, are also possible provided a basicgroup, such as pyridyl, constitutes part of the structure.

The neutral forms of the compounds can be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are eq to the parent form of thecompound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

As used herein, the terms “Porphyromonas gingivalis” and “P. gingivalis”refer to the gram-negative asaccharolytic bacterium that is recognizedas a key causative microbe in the pathogenesis of periodontitis andrelated conditions. “P. gingivalis infection” refers to the invasion andcolonization of P. gingivalis in a bodily tissue such as the gums or thebrain. P. gingivalis infection is frequently characterized by subsequenttissue injury and disease.

As used herein, the term “gingipain” refers to cysteine proteasesexpressed by P. gingivalis having trypsin-like specificity (i.e.,Lys-Xaa and Arg-Xaa). Gingipains are recognized as the major virulencefactors of P. gingivalis and contribute to bacterial attachment andcolonization, nutrient acquisition, evasion of host defenses, and tissueinvasion. The terms “arginine gingipain” and “Rgp” are usedinterchangeably to refer to the P. gingivalis arginine-specificgingipains RgpA and RgpB, classified under EC number EC 3.4.22.37. ThergpA and rgpB gene-translation products, RgpA and RgpB, share acaspase-like protease domain (specific for Arg-Xaa peptide bonds) and animmunoglobulin-like domain. In RgpA, the protease andimmunoglobulin-like domains are followed by a large C-terminal extensioncontaining hemagglutinin-adhesin domains.

As used herein, the term “inhibiting” refers to reducing the level ofactivity (e.g., proteolytic activity) of an enzyme such as a gingipainwhich can be assessed, for example, using an in vitro assay or othersuitable assay. Inhibition of enzyme activity caused by a particularsubstance (e.g., a gingipain inhibitor as described herein) can beexpressed as the percentage of the enzyme activity measured in theabsence of the substance under similar conditions. The ability of aparticular substance to inhibit an enzyme can be expressed as an IC₅₀value, i.e., the concentration of the compound required to reduce theactivity of the enzyme to 50% of its maximum activity.

As used herein, the terms “treat,” “treatment,” and “treating” refer toany indicia of success in the treatment or amelioration of an injury,pathology, condition, or symptom (e.g., cognitive impairment), includingany objective or subjective parameter such as abatement; remission;diminishing of symptoms or making the symptom, injury, pathology orcondition more tolerable to the patient; reduction in the rate ofsymptom progression; decreasing the frequency or duration of the symptomor condition; or, in some situations, preventing the onset of thesymptom. The treatment or amelioration of symptoms can be based on anyobjective or subjective parameter, including, e.g., the result of aphysical examination.

As used herein, the terms “effective amount” and “therapeuticallyeffective amount” refer to a dose of a compound such as an Rgp inhibitorthat inhibits the activity of a gingipain and/or produces therapeuticeffects for which it is administered. The exact dose will depend on thepurpose of the treatment, and will be ascertainable by one skilled inthe art using known techniques (see, e.g., Lieberman, PharmaceuticalDosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technologyof Pharmaceutical Compounding (1999); Pickar, Dosage Calculations(1999); Goodman & Gilman's The Pharmacological Basis of Therapeutics,11^(th) Edition, 2006, Brunton, Ed., McGraw-Hill; and Remington: TheScience and Practice of Pharmacy, 21^(st) Edition, 2005, Hendrickson,Ed., Lippincott, Williams & Wilkins).

As used herein, the term “Alzheimer's disease” refers to a progressivedisease of the central nervous system in humans and other mammals. It ismanifested by dementia (especially in the elderly); disorientation; lossof memory; difficulty with language, calculation, or visual-spatialskills; and psychiatric manifestations. Alzheimer's disease isassociated with progressive neurodegeneration and characteristicpathology, namely beta amyloid plaques and tau tangles.

As used herein, the term “osteoarthritis” refers to a chronicdegenerative joint disease that results from breakdown of jointcartilage, synovial tissue, and underlying bone.

As used herein, the term “subject” refers to animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like.

III. Gingipain Inhibitors

In one embodiment, the invention provides a compound according toFormula I:

-   -   or a pharmaceutically acceptable salt thereof, wherein    -   W is selected from a bond, CH₂, and O;    -   R^(1a) and R^(1b) are independently selected from H and C₁₋₆        alkyl;    -   R² is selected from C₁₋₆ alkyl and halogen;    -   subscript n is 0 or 1;    -   R³ is selected from C₃₋₈ alkyl, C₃₋₈ cycloalkyl, C₃₋₁₂        heterocyclyl, C₆₋₁₀ aryl, and C₅₋₁₂ heteroaryl wherein R³ is        optionally substituted with one or more R^(3a) substituents;    -   each R^(3a) is independently selected from halogen, —CN, —NO₂,        —N₃, —OH, C₁₋₄ alkyl, C₁₋₄ haloalkyl, C₁₋₄ alkoxy,        -   C₁₋₄ haloalkoxy, —N(R^(c))₂, —(CH₂)_(k)C(O)R^(b),            —NR^(c)(CH₂)_(u)C(O)R^(b), —O(CH₂)_(u)C(O)R^(b),            —(CH₂)_(k)CONR^(c)R^(c), —(CH₂)_(k)NR^(c)C(O)R^(b),        -   —NR^(c)(CH₂)_(u)CONR^(c)R^(c),            —NR^(c)(CH₂)_(u)NR^(c)C—O)R^(b), —O(CH₂)_(u)CONR^(c)R^(c),        -   and —O(CH₂)_(u)NR^(c)C(O)R^(b), and optionally substituted            triazolyl;    -   each R^(b) is independently selected from C₁₋₄ alkyl, C₁₋₄        haloalkyl, and C₁₋₄ deuteroalkyl;    -   each R^(c) is independently selected from hydrogen and C₁₋₈        alkyl;    -   each subscript k is independently selected from 0, 1, 2, 3, 4,        5, and 6;    -   each subscript u is independently selected from 1, 2, 3, 4, 5,        and 6;    -   R⁴ is selected from —CH₂R^(4a) and C₁₋₆ haloalkyl;    -   R^(4a) is selected from —O—R⁵, —S—R⁶, —SO—R⁶, —SO₂—R⁶, —N(R⁷)₂,        and C₅₋₁₂ heteroaryl;    -   R⁵ is selected from phenyl, C₁₋₆ alkyl, C₁₋₆ haloalkyl, and        C₅₋₁₂ heteroaryl,        -   wherein phenyl is substituted with 1-5 halogens, and        -   wherein C₅₋₁₂ heteroaryl is optionally substituted with            halogen or C₁₋₃ haloalkyl;    -   R⁶ is selected from phenyl, C₁₋₆ alkyl, C₁₋₆ haloalkyl, and        C₅₋₁₂ heteroaryl,        -   wherein phenyl is optionally substituted with 1-5 halogens,            and        -   wherein C₅₋₁₂ heteroaryl is optionally substituted with            halogen or C₁₋₃ haloalkyl; and    -   each R⁷ is independently selected C₁₋₆ alkyl.

In some embodiments, each of R^(1a) and R^(1b) is independently selectedfrom H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,isobutyl, tert-butyl, n-pentyl, branched pentyl, n-hexyl, and branchedhexyl. In some embodiments, each of R^(1a) is H and R^(1b) is selectedfrom H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,isobutyl, tert-butyl, n-pentyl, branched pentyl, n-hexyl, and branchedhexyl. In some embodiments, R^(1b) is independently selected from H,methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,tert-butyl, n-pentyl, branched pentyl, n-hexyl, and branched hexyl andR^(1a) is H. In some embodiments, R^(1a) and R^(1b) are H.

Compounds of the invention can be prepared in protected form (e.g.,protected compounds wherein at least one of R^(1a) and R^(1b) is anamine protecting group). A number of suitable protecting groups—asdescribed, for example, by Green and Wuts (Protective Groups in OrganicSynthesis, 4^(th) Ed 2007, Wiley-Interscience, New York)—can be used. Insome embodiments, R^(1a) is H and R^(1b) is selected frombenzyloxycarbonyl; 9-fluorenylmethyl-oxycarbonyl; tert-butyloxycarbonyl; and allyloxycarbonyl. In some embodiments, R^(1a) andR^(1b) are selected from benzyloxycarbonyl;9-fluorenylmethyl-oxycarbonyl; tert-butyl oxycarbonyl; andallyloxycarbonyl. In some embodiments, R^(1a) is H and R^(1b)is/c/7-butyl oxycarbonyl, Compounds can also be prepared in alkylatedform (i.e., compounds wherein at least one of R^(1a) and R^(1b) is analkyl group). One or both of R^(1a) and R^(1b) can be, for example,methyl, ethyl, n-propyl, isopropyl, n-butyl, or t-butyl.

In some embodiments, subscript n is 0. In some embodiments, subscript pis 1. In some embodiments, subscript n is 1 and R² is selected fromfluoro, chloro, bromo, iodo, methyl, ethyl, n-propyl, isopropyl,n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, branched pentyl,n-hexyl, and branched hexyl. In some embodiments, subscript n is 1 andR² is selected from fluoro, chloro, and methyl. In some embodiments,subscript n is 2 or 3.

In some embodiments, W is a bond. In some embodiments, W is selectedfrom CH₂ and O.

In some embodiments, the compound has a structure according to FormulaHa:

In some embodiments, the compound has a structure according to FormulaIIb:

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R³ is selected from C₃₋₈ cycloalkyl, C₆₋₁₀ aryl, C₅₋₁₂heteroaryl, and C₃₋₁₂ heterocyclyl, each of which is optionallysubstituted with one or more R^(3a) substituents. For example, R³ can becyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl. In some embodiments, R³ is selected from cyclobutyl,cyclopentyl, and cyclohexyl. In some embodiments, R³ is selected fromphenyl and naphthyl. In some embodiments, R³ is selected from pyrrolyl,pyridinyl, imidazolyl, pyrazolyl, triazolyl, pyrazinyl, triazinyl,indolyl, isoindolyl, and quinolinyl. In some such embodiments, R³ isselected from cyclopentyl and phenyl, each of which is optionallysubstituted with one or more R^(3a) substituents. In some suchembodiments, each R^(3a) is independently selected from halogen, —N₃,C₁₋₄ alkyl, C₁₋₄ haloalkyl, C₁₋₄ alkoxy, C₁₋₄ haloalkoxy, and—NR^(c)C(O)R^(b). In some embodiments, R³ is cyclopentyl.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R³ is selected from C₃₋₈ cycloalkyl, C₆₋₁₀ aryl, C₅₋₁₂heteroaryl, and C₃₋₁₂ heterocyclyl, each of which is optionallysubstituted with one or more R^(3a) substituents. In some suchembodiments, R³ is selected from cyclopentyl and phenyl, each of whichis optionally substituted with one or more R^(3a) substituents. In somesuch embodiments, each R^(3a) is independently selected from halogen,—N₃, C₁₋₄ alkyl, C₁₋₄ haloalkyl, C₁₋₄ alkoxy, C₁₋₄ haloalkoxy, and—NR^(c)C(O)R^(b). In some embodiments, R³ is cyclopentyl.

One of skill in the art will appreciate that compounds containing azidegroups (e.g., compounds wherein R^(3a) is −N₃) can be modified withfurther functional groups via reaction with a complementary reactionpartner such as an alkyne-bearing compound or a phosphine-bearingcompound. Reaction of azides and alkynes via [3+2] cycloaddition,commonly referred to as “click chemistry,” can be used to install avariety of substituted triazole groups in the compounds of theinvention. Accordingly, some embodiments of the invention providecompounds wherein R^(3a) is an optionally substituted triazolyl moietyaccording to the formula:

wherein R^(3b) is a functional moiety and L³ is a linking moiety.

In some embodiments, the linking moiety L³ has a structure-L^(3a)-L^(3b)-, wherein L^(3a) and L^(3b) are independently selectedfrom a bond, a divalent polymer moiety, and linear or branched,saturated or unsaturated C₁₋₃₀ alkyl; wherein:

-   -   one or more carbon atoms in the C₁₋₃₀ alkyl is optionally and        independently replaced by O, S, NR^(a);    -   two or more groupings of adjacent carbon atoms in the C₁₋₃₀        alkyl are optionally and independently replaced by —NR^(a)(CO)—        or —(CO)NR^(a)—; and    -   two or more groupings of adjacent carbon atoms in the C₁₋₃₀        alkyl are optionally and independently replaced by a 4- to        8-membered, divalent carbocycle or a 4- to 8-membered, divalent        heterocycle having one to four heteroatoms selected from O, S,        and N; and    -   each R^(a) is independently selected from H and C₁₋₆ alkyl.

In some embodiments, the functional group R^(3b) is selected from achromophore, a fluorophore, and a binding moiety (e.g., biotin,glutathione, and the like).

In some embodiments, R³ is selected from C₃₋₈ alkyl and C₃₋₈ cycloalkyl,each of which is optionally substituted with one or more R^(3a)substituents. In some embodiments, R³ is selected from cyclopentyl andisopropyl. In some embodiments, R³ is unsubstituted cyclopentyl. In someembodiments, R³ is isopropyl and R^(3a) is methoxy.

In certain embodiments, R³ and the carbonyl to which it is bonded form amoiety other than a naturally-occurring amino acid residue (an L aminoacid residue) or an isomer of a naturally-occurring amino acid residue(a D amino acid residue). In some embodiments, R³ and the carbonyl towhich it is bonded form a moiety other than asparaginyl, substitutedasparaginyl, glutaminyl (i.e., a glutamine residue), substitutedglutaminyl (i.e., a substituted glutamine residue), glutamyl (i.e., aglutamic acid residue), substituted glutamyl (i.e., a substitutedglutamic acid residue), isoleucinyl, substituted isoleucinyl, leucinyl,substituted leucinyl, lysinyl, substituted lysinyl, methioninyl,substituted methioninyl, prolinyl, substituted prolinyl, threoninyl,substituted threoninyl, valinyl, or substituted valinyl. The substitutedamino acid residues may be present in larger peptide groups having twoor more amino acid residues linked via amine bonds.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is selected from —S—R⁶, —SO—R⁶, —SO₂—R⁶, C₅₋₁₂heteroaryl, and —N—R⁷. In some embodiments, R^(4a) is selected from—O—R⁵, C₅₋₁₂ heteroaryl, and —N—(R⁷)₂. In some embodiments, R^(4a) isselected from —O—R⁵, —S—R⁶, —SO—R⁶, —SO₂—R⁶, and —N—(R⁷)₂. In someembodiments, R^(4a) is selected from —O—R⁵, —S—R⁶, —SO—R⁶, —SO₂—R⁶, andC₅₋₁₂ heteroaryl.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is selected from C₁₋₆ haloalkoxy, C₁₋₆alkylthio, C₁₋₆ haloalkythio, C₁₋₆ alkylsufonyl, (C₁₋₆ dialkyl)amino,C₅₋₁₂ heteroaryl, —O-phenyl wherein phenyl is substituted with 1-5halogens, and —S-phenyl wherein phenyl is optionally substituted with1-5 halogens.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is selected from C₁₋₆ alkoxy, C₁₋₆ alkylthio,C₁₋₆ haloalkythio, C₁₋₆ alkylsufonyl, (C₁₋₆ dialkyl)amino, C₅₋₁₂heteroaryl, —O-phenyl wherein phenyl is substituted with 1-5 halogens,and —S-phenyl wherein phenyl is optionally substituted with 1-5halogens.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is selected from C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,C₁₋₆ haloalkythio, C₁₋₆ alkylsufonyl, (C₁₋₆ dialkyl)amino, C₅₋₁₂heteroaryl, —O-phenyl wherein phenyl is substituted with 1-5 halogens,and —S-phenyl wherein phenyl is optionally substituted with 1-5halogens.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is selected from C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,C₁₋₆ alkylthio, C₁₋₆ alkylsufonyl, (C₁₋₆ dialkyl)amino, C₅₋₁₂heteroaryl, —O-phenyl wherein phenyl is substituted with 1-5 halogens,and —S-phenyl wherein phenyl is optionally substituted with 1-5halogens.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is selected from C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,C₁₋₆ alkylthio, C₁₋₆ haloalkythio, (C₁₋₆ dialkyl)amino, C₅₋₁₂heteroaryl, —O-phenyl wherein phenyl is substituted with 1-5 halogens,and —S-phenyl wherein phenyl is optionally substituted with 1-5halogens.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is selected from C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,C₁₋₆ alkylthio, C₁₋₆ haloalkythio, C₁₋₆ alkylsufonyl, C₅₋₁₂ heteroaryl,—O-phenyl wherein phenyl is substituted with 1-5 halogens, and —S-phenylwherein phenyl is optionally substituted with 1-5 halogens.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is selected from C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,C₁₋₆ alkylthio, C₁₋₆ haloalkythio, C₁₋₆ alkylsufonyl, (C₁₋₆dialkyl)amino, —O-phenyl wherein phenyl is substituted with 1-5halogens, and —S-phenyl wherein phenyl is optionally substituted with1-5 halogens.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is selected from C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,C₁₋₆ alkylthio, C₁₋₆ haloalkythio, C₁₋₆ alkylsufonyl, (C₁₋₆dialkyl)amino, C₅₋₁₂ heteroaryl, and —S-phenyl wherein phenyl isoptionally substituted with 1-5 halogens.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is selected from C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,C₁₋₆ alkylthio, C₁₋₆ haloalkythio, C₁₋₆ alkylsufonyl, (C₁₋₆dialkyl)amino, C₅₋₁₂ heteroaryl, and —O-phenyl wherein phenyl issubstituted with 1-5 halogens.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R⁴ is —CH₂—O—R⁵ and R⁵ is selected from1,1,1,3,3,3-hexafluoroprop-2-yl, isoxazolyl, and phenyl, wherein phenylis substituted with 1-5 halogens.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R⁵ is phenyl substituted with 1-5 halogens. In someembodiments, each halogen in R⁵ is selected from F and Cl. In someembodiments, each halogen in R⁵ is F.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is —OR⁵, such that R⁴ is a moiety having thestructure:

-   -   wherein R^(5a), R^(5b), R^(5c), R^(5d), and R^(5e) are        independently selected from hydrogen and halogen, and the wavy        line represents the point of connection to the compound.

In some embodiments:

-   -   R^(5a) is halogen, R^(5b) is H, R^(5c) is H, R^(5d) is H, and        R^(5e) is H; or    -   R^(5a) is H, R^(5b) is halogen, R^(5c) is H, R^(5d) is H, and        R^(5e) is H; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is halogen, R^(5d) is H, and        R^(5e) is H; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is H, R^(5d) is halogen, and        R^(5e) is H; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is H, R^(5d) is H, and R^(5e)        is halogen; or    -   R^(5a) is halogen, R^(5b) is halogen, R^(5c) is H, R^(5d) is H,        and R^(5e) is H; or    -   R^(5a) is halogen, R^(5b) is H, R^(5c) is halogen, R^(5d) is H,        and R^(5e) is H; or    -   R^(5a) is halogen, R^(5b) is H, R^(5c) is H, R^(5d) is halogen,        and R^(5e) is H; or    -   R^(5a) is halogen, R^(5b) is H, R^(5c) is H, R^(5d) is H, and        R^(5e) is halogen; or    -   R^(5a) is H, R^(5b) is halogen, R^(5c) is halogen, R^(5d) is H,        and R^(5e) is H; or    -   R^(5a) is H, R^(5b) is halogen, R^(5c) is H, R^(5d) is halogen,        and R^(5e) is H; or    -   R^(5a) is H, R^(5b) is halogen, R^(5c) is H, R^(5d) is H, and        R^(5e) is halogen; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is halogen, R^(5d) is halogen,        and R^(5e) is H; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is halogen, R^(5d) is H, and        R^(5e) is halogen; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is H, R^(5d) is halogen, and        R^(5e) is halogen; or    -   R^(5a) is halogen, R^(5b) is halogen, R^(5c) is halogen, R^(5d)        is H, and R^(5e) is H; or    -   R^(5a) is halogen, R^(5b) is halogen, R^(5c) is H, R^(5d) is        halogen, and R^(5e) is H; or    -   R^(5a) is halogen, R^(5b) is halogen, R^(5c) is H, R^(5d) is H,        and R^(5e) is halogen; or    -   R^(5a) is halogen, R^(5b) is H, R^(5c) is halogen, R^(5d) is        halogen, and R^(5e) is H; or    -   R^(5a) is halogen, R^(5b) is H, R^(5c) is halogen, R^(5d) is H,        and R^(5e) is halogen; or    -   R^(5a) is halogen, R^(5b) is H, R^(5c) is H, R^(5d) is halogen,        and R^(5e) is halogen; or    -   R^(5a) is H, R^(5b) is halogen, R^(5c) is halogen, R^(5d) is        halogen, and R^(5e) is H; or    -   R^(5a) is H, R^(5b) is halogen, R^(5c) is halogen, R^(5d) is H,        and R^(5e) is halogen; or    -   R^(5a) is H, R^(5b) is halogen, R^(5c) is H, R^(5d) is halogen,        and R^(5e) is halogen; or    -   R^(5a) is H, R^(5b) is H, R^(5C) is halogen, R^(5d) is halogen,        and R^(5e) is halogen; or    -   R^(5a) is H, R^(5b) is halogen, R^(5c) is halogen, R^(5d) is        halogen, and R^(5e) is halogen; or    -   R^(5a) is halogen, R^(5b) is H, R^(5c) is halogen, R^(5d) is        halogen, and R^(5e) is halogen; or    -   R^(5a) is halogen, R^(5b) is halogen, R^(5c) is halogen, R^(5d)        is H, and R^(5e) is halogen; or    -   R^(5a) is halogen, R^(5b) is halogen, R^(5c) is halogen, R^(5d)        is halogen, and R^(5e) is H; or    -   R^(5a) is halogen, R^(5b) is halogen, R^(5c) is H, R^(5d) is        halogen, and R^(5e) is halogen.

In some embodiments:

-   -   R^(5a) is F or Cl, R^(5b) is H, R^(5c) is H, R^(5d) is H, and        R^(5e) is H; or    -   R^(5a) is H, R^(5b) is F or Cl, R^(5c) is H, R^(5d) is H, and        R^(5e) is H; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is F or Cl, R^(5d) is H, and        R^(5e) is H; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is H, R^(5d) is F or Cl, and        R^(5e) is H; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is H, R^(5d) is H, and R^(5e)        is F or Cl; or    -   R^(5a) is F or Cl, R^(5b) is F or Cl, R^(5c) is H, R^(5d) is H,        and R^(5e) is H; or    -   R^(5a) is F or Cl, R^(5b) is H, R^(5c) is F or Cl, R^(5d) is H,        and R^(5e) is H; or    -   R^(5a) is F or Cl, R^(5b) is H, R^(5c) is H, R^(5d) is F or Cl,        and R^(5e) is H; or    -   R^(5a) is F or Cl, R^(5b) is H, R^(5c) is H, R^(5d) is H, and        R^(5e) is F or Cl; or    -   R^(5a) is H, R^(5b) is F or Cl, R^(5c) is F or Cl, R^(5d) is H,        and R^(5e) is H; or    -   R^(5a) is H, R^(5b) is F or Cl, R^(5c) is H, R^(5d) is F or Cl,        and R^(5e) is H; or    -   R^(5a) is H, R^(5b) is F or Cl, R^(5c) is H, R^(5d) is H, and        R^(5e) is F or Cl; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is F or Cl, R^(5d) is F or Cl,        and R^(5e) is H; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is F or Cl, R^(5d) is H, and        R^(5e) is F or Cl; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is H, R^(5d) is F or Cl, and        R^(5e) is F or Cl; or    -   R^(5a) is F or Cl, R^(5b) is F or Cl, R^(5c) is F or Cl, R^(5d)        is H, and R^(5e) is H; or    -   R^(5a) is F or Cl, R^(5b) is F or Cl, R^(5c) is H, R^(5d) is F        or Cl, and R^(5e) is H; or    -   R^(5a) is F or Cl, R^(5b) is F or Cl, R^(5c) is H, R^(5d) is H,        and R^(5e) is F or Cl; or    -   R^(5a) is F or Cl, R^(5b) is H, R^(5c) is F or Cl, R^(5d) is F        or Cl, and R^(5e) is H; or    -   R^(5a) is F or Cl, R^(5b) is H, R^(5c) is F or Cl, R^(5d) is H,        and R^(5e) is F or Cl; or    -   R^(5a) is F or Cl, R^(5b) is H, R^(5c) is H, R^(5d) is F or Cl,        and R^(5e) is F or Cl; or    -   R^(5a) is H, R^(5b) is F or Cl, R^(5c) is F or Cl, R^(5d) is F        or Cl, and R^(5e) is H; or    -   R^(5a) is H, R^(5b) is F or Cl, R^(5c) is F or Cl, R^(5d) is H,        and R^(5e) is F or Cl; or    -   R^(5a) is H, R^(5b) is F or Cl, R^(5c) is H, R^(5d) is F or Cl,        and R^(5e) is F or Cl; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is F or Cl, R^(5d) is F or Cl,        and R^(5e) is F or Cl; or    -   R^(5a) is H, R^(5b) is F or Cl, R^(5c) is F or Cl, R^(5d) is F        or Cl, and R^(5e) is F or Cl; or    -   R^(5a) is F or Cl, R^(5b) is H, R^(5c) is F or Cl, R^(5d) is F        or Cl, and R^(5e) is F or Cl; or    -   R^(5a) is F or Cl, R^(5b) is F or Cl, R^(5c) is F or Cl, R^(5d)        is H, and R^(5e) is F or Cl; or    -   R^(5a) is F or Cl, R^(5b) is F or Cl, R^(5c) is F or Cl, R^(5d)        is H, and R^(5e) is F or Cl; or    -   R^(5a) is F or Cl, R^(5b) is F or Cl, R^(5c) is H, R^(5d) is F        or Cl, and R^(5e) is F or Cl.

In some embodiments:

-   -   R^(5a) is F, R^(5b) is H, R^(5c) is H, R^(5d) is H, and R^(5e)        is H; or    -   R^(5a) is H, R^(5b) is F, R^(5c) is H, R^(5d) is H, and R^(5e)        is H; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is F, R^(5d) is H, and R^(5e)        is H; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is H, R^(5d) is F, and R^(5e)        is H; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is H, R^(5d) is H, and R^(5e)        is F; or    -   R^(5a) is F, R^(5b) is F, R^(5c) is H, R^(5d) is H, and R^(5e)        is H; or    -   R^(5a) is F, R^(5b) is H, R^(5c) is F, R^(5d) is H, and R^(5e)        is H; or    -   R^(5a) is F, R^(5b) is H, R^(5c) is H, R^(5d) is F, and R^(5e)        is H; or    -   R^(5a) is F, R^(5b) is H, R^(5c) is H, R^(5d) is H, and R^(5e)        is F; or    -   R^(5a) is H, R^(5b) is F, R^(5c) is F, R^(5d) is H, and R^(5e)        is H; or    -   R^(5a) is H, R^(5b) is F, R^(5c) is H, R^(5d) is F, and R^(5e)        is H; or    -   R^(5a) is H, R^(5b) is F, R^(5c) is H, R^(5d) is H, and R^(5e)        is F; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is F, R^(5d) is F, and R^(5e)        is H; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is F, R^(5d) is H, and R^(5e)        is F; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is H, R^(5d) is F, and R^(5e)        is F; or    -   R^(5a) is F, R^(5b) is F, R^(5c) is F, R^(5d) is H, and R^(5e)        is H; or    -   R^(5a) is F, R^(5b) is F, R^(5c) is H, R^(5d) is F, and R^(5e)        is H; or    -   R^(5a) is F, R^(5b) is F, R^(5c) is H, R^(5d) is H, and R^(5e)        is F; or    -   R^(5a) is F, R^(5b) is H, R^(5c) is F, R^(5d) is F, and R^(5e)        is H; or    -   R^(5a) is F, R^(5b) is H, R^(5c) is F, R^(5d) is H, and R^(5e)        is F; or    -   R^(5a) is F, R^(5b) is H, R^(5c) is H, R^(5d) is F, and R^(5e)        is F; or    -   R^(5a) is H, R^(5b) is F, R^(5c) is F, R^(5d) is F, and R^(5e)        is H; or    -   R^(5a) is H, R^(5b) is F, R^(5c) is F, R^(5d) is H, and R^(5e)        is F; or    -   R^(5a) is H, R^(5b) is F, R^(5c) is H, R^(5d) is F, and R^(5e)        is F; or    -   R^(5a) is H, R^(5b) is H, R^(5c) is F, R^(5d) is F, and R^(5e)        is F; or    -   R^(5a) is H, R^(5b) is F, R^(5c) is F, R^(5d) is F, and R^(5e)        is F; or    -   R^(5a) is F, R^(5b) is H, R^(5c) is F, R^(5d) is F, and R^(5e)        is F; or    -   R^(5a) is F, R^(5b) is F, R^(5c) is F, R^(5d) is H, and R^(5e)        is F; or    -   R^(5a) is F, R^(5b) is F, R^(5c) is F, R^(5d) is H, and R^(5e)        is F; or    -   R^(5a) is F, R^(5b) is F, R^(5c) is H, R^(5d) is F, and R^(5e)        is F.

In certain embodiments, R⁴ is not 2,3,5,6-tetrafluorophenoxymethyl.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R⁵ is selected from 2-fluorophenyl; 3-fluorophenyl;4-fluorophenyl; 2,3-difluorophenyl; 2,4-difluorophenyl;2,5-difluorophenyl; 2,6-difluorophenyl; 3,4-difluorophenyl;3,5-difluorophenyl; 2,3,4-trifluorophenyl; 3,4,5-trifluorophenyl;2,3,6-trifluorophenyl; 2,3,5-trifluorophenyl; and2,3,5,6-tetrafluorophenyl.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R⁵ is selected from 2,6-difluorophenyl;2,3,6-trifluorophenyl; and 2,3,5,6-tetrafluorophenyl.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is C₅₋₁₂ heteroaryl. R^(4a) can be, for example,pyrrolyl, pyridinyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,pyrazinyl, triazinyl, indolyl, isoindolyl, or quinolinyl. In someembodiments, R^(4a) is isoxazolyl.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is —O—R⁵, wherein R⁵ is C₁₋₆ haloalkyl. In suchembodiments, R⁵ can be, e.g., chloromethyl, dichloromethyl,trichloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl,2,2,2-trichloroethyl, 2,2,2-trifluoroethyl, pentachloroethyl,pentafluoroethyl, 1,1,1,3,3,3-hexachloropropyl,1,1,1,3,3,3-hexafluoropropyl, or the like. In some embodiments, R⁵ is1,1,1,3,3,3-hexafluoroprop-2-yl.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is selected from —S—R⁶ and —SO₂—R⁶, wherein R⁶is C₁₋₆ alkyl. In such embodiments, R⁶ can be, e.g., methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec butyl, tert butyl, pentyl,isopentyl, or hexyl.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R^(4a) is —N(R⁷)₂, wherein each R⁷ is independentlyselected C₁₋₆ alkyl. In such embodiments, each R⁷ can independently be,e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec butyl, tertbutyl, pentyl, isopentyl, or hexyl.

In some embodiments, the invention provides compounds of Formula I,Formula Ha, and/or Formula IIb, and pharmaceutically acceptable saltsthereof, wherein R⁴ is haloalkyl. For example, R⁵ can be chloromethyl,dichloromethyl, trichloromethyl, difluoromethyl, or trifluoromethyl.

The compounds of the invention can be further substituted; a compoundaccording to Formula I may contain, for example, an optionallysubstituted R^(1a) and/or R^(1b) group, one or more optionallysubstituted R² groups, an optionally substituted R³ group, and/or anoptionally substituted R⁴ group (including, an optionally substituted R⁵group, an optionally substituted R⁶ group, and/or one or more optionallysubstituted R⁷ groups).

In some embodiments, the aminopyridyl ketone compound is selected from:

and pharmaceutically acceptable salts thereof.

Aminopyridyl ketones may be prepared by the following approaches,summarized in Scheme 1 and described below. As shown in Scheme 1,aminopyridines (e.g., 2-amino-4-formylpyridine and2-amino-5-formylpyridine (xi)) can be reacted with a suitable protectingreagent (e.g., di-tert-butyl dicarbonate) to prepare the correspondingmono- and/or di-protected formylpyridines (xii), wherein P¹ is H or aprotecting group and P² is a protecting group. Any of these aldehydes(xii) may be reacted with a protected phosphonate (xiii) (wherein Q¹ isH or a protecting group; Q² is a protecting group; and each R isindependently C₁₋₆ alkyl; e.g., ZHNCH(CO₂Me)PO₃Me₂) and a strong base toprepare dehydro-aminopyridylalanine methyl esters (xiv).

When Q¹ and/or Q² is a Z group (or another protecting group which can beremoved by hydrogenation), dehydro-aminopyridylalanines (xiv) may behydrogenated in the presence of palladium on carbon, providingaminoesters (xv) by simultaneously saturating the olefins anddeprotecting the α-amino group. The free amino groups may be reactedwith carboxylic acids (R³C(O)OH) and dehydrating agents to formamidoesters (xvi). The dehydrating agent may be HATU or any of manyother reagents suitable for carboxamide formation. The amidoesters maybe converted to protected products (xvii) by various routes. In onenon-limiting example, the amidoesters are hydrolyzed using a strongbased such as NaOH. The resulting carboxylic acids are then reacted withClCO₂Et, a tertiary amine, and diazo methane to form diazomethylketones, which can then be treated with HBr to give bromomethyl ketones.In another sequence, the methyl esters may be treated with ClCH₂I andLiN(iPr)₂ to provide chloromethyl ketones in one step. The bromomethylketones or chloromethyl ketones can be heated with substituted phenolsand KF in DMF to give aryloxymethyl ketones. In another non-limitingexample, the bromomethyl or chloromethyl ketones are treated withisoxazole-5-one and KF in DMF to give isoxazolyloxymethyl ketones.Lastly, the protecting groups P¹ and P² (e.g., Boc groups) may beremoved under suitable conditions (e.g., via treatment withtrifluoroacetic acid), to afford the desired final products.

The starting materials and reagents used in preparing the compounds ofthe invention are either available from commercial suppliers or areprepared by methods known to those skilled in the art followingprocedures set forth in references such as Fieser and Fieser's Reagentsfor Organic Synthesis, Vol. 1-28 (Wiley, 2016); March's Advanced OrganicChemistry, 7^(th) Ed. (Wiley, 2013); and Larock's Comprehensive OrganicTransformations, 2^(nd) Ed. (Wiley, 1999). The starting materials andthe intermediates of the reaction can be isolated and purified ifdesired using conventional techniques including, but not limited to,filtration, distillation, crystallization, chromatography and the like.Such materials can be characterized using conventional means, includingmeasuring physical constants and obtaining spectral data.

Unless specified to the contrary, the reactions described herein takeplace at atmospheric pressure over a temperature range of from about−78° C. to about 250° C. For example, reactions can be conducted at fromabout 0° C. to about 125° C., or at about room (or ambient) temperature,e.g., about 20° C. In some embodiments, reactions are conducted at about0° C., 20° C., 25° C., 90° C., 100° C., 110° C., 125° C., 150° C., 175°C., or 200° C. In some embodiments, reactions are conducted starting ata first temperature (e.g., about −78° C. or about 0° C.), and allowed towarm to a higher second temperature (e.g., about 20° C. or about 25°C.). One of skill in the art will appreciate that various modificationsto the procedures described herein can be made.

The invention provides a number of useful aminopyridinecyanamidecompounds having various aminopyridine moieties such as aminopyridines(e.g., 2-aminopyridines), aminoisoquinolines (e.g., 1-aminoisoquinolinesand 1-amino-6,7,8,9-tetrahydrobenzo[g]iso-quinolines),aminofuropyridines (e.g., 7-aminofuro[2,3-c]pyridines and4-aminofuro[3,2-c]pyridines), and aminonapthyridines (e.g.,3-amino-1,7-naphthyridines and 8-amino-2,7-naphthyridines).

In one embodiment, the invention provides a compound according toFormula III:

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   R¹¹ is selected from C₁₋₆ alkyl and C₃₋₈ cycloalkyl;    -   R^(12a) and R^(12b) are independently selected from H, C₁₋₆        alkyl, and C₆₋₁₀ aryl, or        -   R^(12a) and R^(12b) are taken together to form C₃₋₆            cycloalkyl, or        -   R^(12a) and R¹¹ are taken together to form 4- to 10-membered            heterocyclyl which is optionally substituted with one or            more R¹⁷;    -   each R^(13a) and each R^(13b) is independently selected from H,        —OH, and C₁₋₆ alkyl, or        -   one R^(13a) and R¹¹ are taken together to form 4- to            10-membered heterocyclyl, or        -   one R^(13b) and R^(12b) are taken together to form a 5- or            6-membered ring;    -   R¹⁴ is selected from H and halogen, or        -   R¹⁴, R^(12a), and R^(12b) are taken together to form a 6- to            8-membered ring, which is optionally substituted with one or            more R¹⁸, or        -   R¹⁴ and one R^(13a) are taken together to form a 5- to            8-membered ring, which is optionally substituted with one or            more R¹⁸, or        -   R¹⁴ is taken together with one R^(13a) and one R^(13b) on            the same carbon atom to form a 5- to 8-membered ring, which            is optionally substituted with one or more R¹⁸, or        -   R¹⁴, R¹¹, and R^(12a) are taken together to form a 6- to            10-membered bicyclic ring, which is optionally substituted            with one or more R¹⁸;    -   R^(15a) and R^(15b) are independently selected from H and C₁₋₆        alkyl;    -   R¹⁶ is independently selected from C₁₋₆ alkyl and halogen;    -   each R¹⁷ is independently selected from C₁₋₆ alkyl, C₁₋₆ alkoxy,        C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, —OH, and —N(R^(17a))₂, wherein        each R^(17a) is independently selected from H and C₁₋₆ alkyl;    -   each R¹⁸ is independently selected from C₁₋₆ alkyl and halogen;    -   Y is selected from O, S, C(R^(19a))₂, and NR^(19b);    -   each R^(19a) is selected from H and C₁₋₆ alkyl, or        -   one R^(19a) and one R^(13b) on adjacent atoms are taken            together to form a double bond;    -   R^(19b) is selected from H and C₁₋₆ alkyl, or        -   R^(19b) and R¹¹ are taken together to form a 4- to            6-membered ring;    -   subscript m is 0, 1, 2, or 3; and    -   subscript q is 0 or 1.

In some embodiments, the invention provides compounds according toFormula IVa:

In some embodiments, the invention provides compounds according toFormula IVb:

In some embodiments, the invention provides compounds according toFormula IVc:

In some embodiments, the invention provides compounds according toFormula V:

In some embodiments, the invention provides compounds according toFormula III or Formula V wherein Y is O or S. In some embodiments, theinvention provides compounds according to Formula III or Formula Vwherein Y is CH₂. In some embodiments, W is selected from O, S, and CH₂,and each R^(13a) and each R^(13b) is selected from H and C₁₋₆ alkyl.

In some embodiments, Y is O and each R^(13a) and each R^(13b) isindependently selected from H, —OH, methyl, ethyl, n-propyl, isopropyl,n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, branched pentyl,n-hexyl, and branched hexyl. In some embodiments, Y is S and eachR^(13a) and each R^(13b) is independently selected from H, —OH, methyl,ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl,n-pentyl, branched pentyl, n-hexyl, and branched hexyl. In someembodiments, Y is CH₂ and each R^(13a) and each R^(13b) is independentlyselected from H, —OH, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, n-pentyl, branched pentyl, n-hexyl, andbranched hexyl.

In some embodiments, Y is selected from O, S, and CH₂, and one ofR^(13a) and R^(13b) is selected from H and —OH, and the remaining13^(3a) and R^(13b) groups are selected from H and C₁₋₆ alkyl.

In some embodiments, Y is CH(R^(19a)), and R^(19a) and one R^(13b) aretaken together to form a double bond. In some embodiments, the grouping—CH(R^(19a))(CR^(13a)R^(13b))_(m)— is selected from ethen-diyl,prop-1-en-1,3-diyl, and but-1-en-1,4-diyl.

In some embodiments, Y is CH(R^(19a)), subscript m is 1, and R^(19a) andR^(13b) are taken together to form a double bond. In some suchembodiments, R^(13a) is selected from H, —OH, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, branchedpentyl, n-hexyl, and branched hexyl H. In some such embodiments, R¹³ isH.

In some embodiments, Y is NR^(19b), and R^(19b) and R¹¹ are takentogether to form a 5- or 6-membered ring. For example, R^(19b) and R¹¹can form imidazolidin-diyl or piperazin-diyl. In some embodiments,R^(19b) and R¹¹ are taken together to form 1,4-piperazin-diyl.

In some embodiments, R^(12a) and R¹¹ are taken together to form 4- to10-membered heterocyclyl in the compounds according to Formula III,Formula IVa, Formula IVb, Formula IVc, or Formula V. For example,R^(12a) and R¹¹ can be taken together to form aziridin-diyl,azetidin-diyl, diazetidin-diyl, pyrrolidin-diyl, imidazolidin-diyl,pyrazolidin-diyl, piperidin-diyl, piperazin-diyl, morpholin-diyl,azepan-diyl, diazepan-diyl, azocan-diyl, or indolin-diyl. In someembodiments, R^(12a) and R¹¹ are taken together to formazetidin-1,2-diyl, pyrrolidin-1,2-diyl, piperidin-1,2-diyl,indolin-1,2-diyl, or isoindolin-1,2-diyl.

In some embodiments, the compound has a structure according to FormulaIII, Formula IVa, Formula IVb, Formula IVc, or Formula V, and R^(12a)and R¹¹ are taken together to form pyrrolidin-1,2-diyl orpiperidin-1,2-diyl. In some embodiments, the pyrrolidin-1,2-diyl orpiperidin-1,2-diyl is substituted with one or two R¹⁷. In someembodiments, the pyrrolidin-1,2-diyl or piperidin-1,2-diyl issubstituted with one R¹⁷. In some embodiments, R¹⁷ is selected from C₁₋₆alkoxy, C₁₋₆ haloalkoxy, —OH, and —N(R^(17a))₂. In some embodiments, R¹⁷is selected from —OH, —NH₂, methoxy, and dimethylamino.

In some embodiments, the compound has a structure according to FormulaV, and R^(12a) and R¹¹ are taken together to form azetidin-1,2-diyl,pyrrolidin-1,2-diyl, piperidin-1,2-diyl, indolin-1,2-diyl, orisoindolin-1,2-diyl. In some embodiments, the azetidin-1,2-diyl,pyrrolidin-1,2-diyl, piperidin-1,2-diyl, indolin-1,2-diyl, orisoindolin-1,2-diyl is substituted with one or two R⁷. In someembodiments, the azetidin-1,2-diyl, pyrrolidin-1,2-diyl,piperidin-1,2-diyl, indolin-1,2-diyl, or isoindolin-1,2-diyl issubstituted with one R¹⁷. In some embodiments, R¹⁷ is C₁₋₆ alkoxy, C₁₋₆haloalkoxy, —OH, or —N(R^(17a))₂. In some embodiments, R¹⁷ is selectedfrom —OH, —NH₂, methoxy, and dimethylamino.

In some embodiments, the invention provides compounds according FormulaIII, Formula IVa, Formula IVb, Formula IVc, and/or Formula V, whereinR^(12a) is H and R^(12b) is selected from H, C₁₋₆ alkyl, and C₆₋₁₀ aryl.For example, R^(12b) can be methyl, ethyl, n-propyl, isopropyl, n-butyl,tert-butyl, or phenyl.

In some embodiments, the invention provides compounds according toFormula III, Formula IVa, Formula IVb, Formula IVc, and/or Formula V,wherein R¹¹ is C₁₋₆ alkyl. For example, R¹¹ can be methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl,branched pentyl, n-hexyl, or branched hexyl. In some embodiments, R¹¹ isC₃₋₈ cycloalkyl. In some embodiments, R¹¹ is cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl. In someembodiments, the invention provides compounds of Formula III, FormulaIVa, Formula IVb, Formula IVc, or Formula V as described above, whereinR¹¹ is selected from methyl, ethyl, isopropyl, cyclopentyl, andcyclohexyl. In some such embodiments, R¹¹ is selected from methyl,isopropyl, and cyclohexyl.

In some embodiments, the invention provides compounds according toFormula III, Formula IVa, Formula IVb, Formula IVc, and/or Formula V,wherein each of R^(15a) and R^(15b) is independently selected from H,methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,tert-butyl, n-pentyl, branched pentyl, n-hexyl, and branched hexyl. Insome embodiments, R^(15a) is H and R^(15b) is selected from H, methyl,ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl,n-pentyl, branched pentyl, n-hexyl, and branched hexyl. In someembodiments, R^(15b) is selected from H, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, branchedpentyl, n-hexyl, and branched hexyl and R^(15a) is H. In someembodiments, R^(15a) and R^(15b) are H.

The aminopyridinecyanamide compounds of the invention can be prepared inprotected form (e.g., protected compounds wherein at least one ofR^(15a), R^(15b), R^(17a), and R^(19b) is an amine protecting group). Anumber of suitable protecting groups—as described, for example, by Greenand Wuts (Protective Groups in Organic Synthesis, 4^(th) Ed 2007,Wiley-Interscience, New York)—can be used. In some embodiments, R^(15a)is H and R^(15b) is selected from benzyloxycarbonyl;9-fluorenylmethyl-oxycarbonyl; tert-butyloxy carbonyl; andallyloxycarbonyl. In some embodiments, R^(15a) and R^(15b) are selectedfrom benzyloxycarbonyl; 9-fluorenylmethyl-oxycarbonyl; tert-butyloxycarbonyl; and allyloxy carbonyl. In some embodiments, R^(15a) is H andR^(15b) is/c/7-butyloxy carbonyl, Compounds can also be prepared inalkylated form (i.e., compounds wherein at least one of R^(15a),R^(15b), R^(17a), and R^(19b) is an alkyl group). One or both of R^(17a)and R^(19b) can be, for example, methyl, ethyl, n-propyl, isopropyl,n-butyl, or t-butyl.

In some embodiments, the invention provides compounds according toFormula III, Formula IVa, Formula IVb, Formula IVc, and/or Formula V,wherein subscript q is 0. In some embodiments, the invention providescompounds according to Formula III, Formula IVa, Formula IVb, FormulaIVc, and/or Formula V, wherein subscript q is 1. In some suchembodiments, subscript q is 1 and R¹⁶ is selected from fluoro, chloro,bromo, iodo, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,isobutyl, tert-butyl, n-pentyl, branched pentyl, n-hexyl, and branchedhexyl. In some embodiments, subscript q is 1 and R¹⁶ is selected fromfluoro, chloro, and methyl. In some embodiments, subscript q is 2.

The compounds of the invention can be further substituted; a compoundaccording to Formula III may contain, for example, an optionallysubstituted R¹¹ group, an optionally substituted R^(12a) and/or R^(12b)group, one or more optionally substituted R^(13a) and/or R^(13b) groups,an optionally substituted R¹⁴ group, an optionally substituted R^(15a)and/or R^(15b) group, one or more optionally substituted R¹⁶ groups, oneor more optionally substituted R¹⁷ groups, one or more optionallysubstituted R¹⁸ groups, one or more optionally substituted R^(19a)groups, and/or an optionally substituted R^(19b) group.

In some embodiments, the aminopyridinecyanamide compound is selectedfrom:

and pharmaceutically acceptable salts thereof.

In some embodiments, the aminopyridinecyanamide compound is selectedfrom:

and pharmaceutically acceptable salts thereof.

In some embodiments, the aminopyridinecyanamide compound is selectedfrom:

and pharmaceutically acceptable salts thereof.

The aminopyridinecyanamide compounds of the invention may be prepared bythe approaches summarized in Scheme 2 and described below. As shown inScheme 2, an amine (i), bearing a reactive group G¹, can be reacted withan amino-heterocycle (ii), bearing a reactive group G². This reactioninvolves transformation of reactive groups G¹ and G² to formintermediate (iii) containing a linkage Y between the amine and theamino-heterocycle structures. Prior to this reaction, the amine isusually protected and the amino-heterocycle is sometimes protected. Ingeneral, P¹¹ is selected from R¹¹ as described above, hydrogen, and aprotecting group; P¹² is selected from hydrogen and a protecting group;and Q¹¹ and Q¹² are independently selected from hydrogen, R^(15a) asdescribed above, R^(15b) as described above, and a protecting group. Anysuitable protecting group (e.g., tert-butoxy carbonyl (Boc),benzyloxycarbonyl (Z), trifluoroacetyl, or another easily removed group)can be used for protecting the amine and the amino-heterocycle, and theprotecting groups are typically chosen so that either may be removedwithout removing the other. For example, Boc may be removed by treatmentwith trifluoroacetic acid, which does not remove Z. Meanwhile, Z may beremoved by hydrogenolysis in the presence of palladium on carbon, whichdoes not remove Boc.

A wide variety of reactive groups G¹ and G² are suitable for forming thelinkage Y between the amine and amino-heterocycle structures. A halideG¹ group or a sulfonate ester G¹ group can undergo a Williamson reactionwith a phenol G² group or thiophenol G² group on the amino-heterocycleto form a linking ether or thioether. Employing a phosphine (e.g., Ph₃P)and an azodicarboxylate (e.g., DEAD) as co-reagents, an alcohol G¹ groupcan undergo a Mitsunobu reaction with a phenol G² group or thiophenol G²group to form a linking ether

or thioether. An unprotected primary amine G¹ group or unprotectedsecondary amine G¹ group can undergo an Ullmann reaction with a halideG² group to form a secondary or tertiary linking amine. The Ullmannreaction can be catalyzed with a homogeneous copper or palladiumcatalyst, as described by Buchwald, Hartwig, Fu, and others.

An olefin G¹ group can undergo a Heck reaction with a halide G² group toform a linking olefin. An alkyne G¹ group can undergo a Sonagashirareaction with a halide G² group to form a linking alkyne. A boronic acidG¹ group can undergo a Suzuki reaction with a halide G² group ortriflate G² group to form a biaryl linkage. A stannane G¹ group canundergo a Stille reaction with a halide G² group or triflate G² group toform a biaryl linkage. After a Heck, Sonagashira, Suzuki, or Stillereaction, certain intermediates (iii) (e.g., intermediates wherein P¹¹and R^(12a) are taken together to form unsaturated heterocyclyl) can behydrogenated to achieve partial or full saturation.

An aldehyde G¹ group on the amine may undergo a Grignard or“Grignard-like” reaction with an organomagnesium or organolithium G²group on the amino-heterocycle, to form a linkage bearing a secondaryalcohol. A carboxylic acid G¹ group on the amine may undergo adecarboxylating photoreaction with a halide G² group on theamino-heterocycle to form a carbon-carbon bond between thedecarboxylated amine and the amino-heterocycle.

After the reaction involving reactive groups G¹ and G², and formation ofthe linkage Y between the amine and amino-heterocycle, the protectinggroup on the amine can be removed to provide an amine (iv), and theamine can be reacted with cyanogen bromide to form a cyanamide (v).After formation of the cyanamide, any protecting group on theamino-heterocycle can be removed to provide products according toFormula III.

Other methods can also be used for preparing an amine (iv) for thecorresponding cyanamide (v). In some instances, the amine precursor canbe obtained via reductive amination of a ketone or aldehyde on theamino-heterocycle. In some instances, the amine precursor can beobtained via reduction of a nitro group on the amino-heterocycle andsubsequent mono-alkylation of the resulting amino G² group. In someinstances, the amine precursor can be obtained via reduction of a cyanogroup on the amino-heterocycle and subsequent monoalkylation of theresulting amino G² group. In some instances, the amine precursor can beobtained via simultaneous reduction of a nitro group and an olefin onthe amino-heterocycle, and subsequent mono-alkylation of the resultingamino G² group. In some instances, the amine precursor can be obtainedvia simultaneous reduction of a cyano group and an olefin on theamino-heterocycle, and subsequent mono-alkylation of the resulting aminoG² group. In some instances, the amine precursor can be obtained via aSchmidt reaction of a carboxylic acid on the amino-heterocycle, andsubsequent mono-alkylation of the resulting amino G² group.

The compounds of the invention are highly active Rgp inhibitors,typically exhibiting Rgp IC₅₀ values in the nanomolar and micromolarrange.

The term “IC₅₀” indicates how much of a compound is needed to inhibit agiven biological process (or component of a process, e.g., an enzyme,cell, cell receptor, or microorganism) by one half (50%). The IC₅₀ valuefor a particular test compound can be measured as follows. Fiftymicroliters (μL) of an enzyme such as RgpA or RgpB (1 nM in 50 mMbis-Tris propane [pH 8.0] containing 1% [vol/vol] Triton X-100 and 5 mM2-mercaptoethanol) is added to columns 1 to 11 of a 96-well plate, and100 μL is added to column 12. Two μL of the test compound (100 μL in100% DMSO) is added to column 12, and the sample is mixed three times bypipetting. Then, a doubling dilution is prepared across the plate byserial transfer into adjacent wells. 50 μL ofZ-Arg-7-amido-4-methylcoumarin (“Z-Arg-AMC;” 40 μM in buffer) is addedto all wells, and the contents are mixed. The reaction is monitored forAMC fluorescence for 15 min at 25° C., and the progress curves areautomatically converted to rates by the Fluoroskan Ascent software. TheIC₅₀ of a compound can then be determined by constructing adose-response curve and examining the effect of different concentrationsof the compound on reversing the activity of the enzyme. From thedose-response curve, IC₅₀ values can be calculated for a given compoundby determining the concentration needed to inhibit half of the maximumbiological response of the enzyme.

The method can also be used to assay enzymes including Kgp, trypsin, andcathepsin B. For Kgp, the substrate can be succinyl-Ala-Phe-Lys-AMC. Fortrypsin, the buffer can contain 10 mM Tris and 10 mM CaCl₂ (pH 8.0), andthe substrate can be Z-Gly-Gly-Arg-AMC. For cathepsin B, the buffer cancontain 50 mM sodium phosphate, 1 mM EDTA, and 10 mM 2-mercaptoethanol(pH 6.25), and the substrate can be Z-Arg-Arg-AMC.

In general, the Rgp IC₅₀ values for compounds of the invention rangefrom about 0.01 nM to about 100 μM. The Rgp IC₅₀ value for a compound ofthe invention can range, for example, from about 0.01 nM to about 0.1nM, or from about 0.1 nM to about 1 nM, or from about 1 nM to about 100nM, or from about 100 nM to about 250 nM, or from about 250 nM to about500 nM, or from about 500 nM to about 750 nM, or from about 750 nM toabout 1 μM, or from about 1 μM to about 10 μM, or from about 10 μM toabout 25 μM, or from about 25 μM to about 50 μM, or from about 50 μM toabout 75 μM, or from about 75 μM to about 100 μM. The Rgp IC₅₀ value fora compound of the invention can range from about 0.01 nM to about 1 nM,or from about 0.05 nM to about 0.75 nM, or from about 0.1 nM to about0.5 nM, from about 1 nM to about 100 nM, or from about 20 nM to about 80nM, or from about 40 nM to about 60 nM, or from about 1 μM to about 100μM, or from about 20 μM to about 80 μM, or from about 40 μM to about 60μM.

In some embodiments, an Rgp inhibitor according to the invention has anRgpB IC₅₀ of 75 nM or less. In some embodiments, the Rgp inhibitor hasan RgpB IC₅₀ of 50 nM or less. In some embodiments, the Rgp inhibitorhas an RgpB IC₅₀ of 25 nM or less. In some embodiments, the Rgpinhibitor has an RgpB IC₅₀ of 10 nM or less. In some embodiments, theRgp inhibitor has an RgpB IC₅₀ of 1 nM or less.

In certain embodiments, Rgp inhibitors according to the invention areselective for Rgp. As used herein, a “selective” Rgp inhibitor is acompound that does not substantially affect the activity of proteasesother than RgpA and RgpB when administered at a therapeuticallyeffective dose for treating a disease or condition associated with P.gingivalis infection. Typically, a protease that is not substantiallyaffected by a particular compound exhibits at least 90% of its normalenzymatic activity in the presence of the compound under physiologicalconditions. Selective Rgp inhibitors include those compounds that do notaffect the activity of proteases other than Rgp when administered at atherapeutically effective dose for treating a brain disorder,periodontal disease, diabetes, a cardiovascular disease, arthritis,rheumatoid arthritis, osteoarthritis, infectious arthritis, psoriaticarthritis, preterm birth, pneumonia, cancer, a kidney disease, a liverdisease, a retinal disorder, or glaucoma associated with P. gingivalisinfection. Preferably, selective Rgp inhibitors do not adversely affectthe coagulation cascade when administered at therapeutically effectivelevels.

IV. Pharmaceutical Compositions and Administration of GingipainInhibitors

In a related embodiment, the invention provides a pharmaceuticalcomposition comprising a compound of Formula I, Formula Ha, Formula IIb,Formula III, Formula IVa, Formula IVb, Formula IVc, or Formula V, and apharmaceutically acceptable excipient. The pharmaceutical compositionscan be prepared by any of the methods well known in the art of pharmacyand drug delivery. In general, methods of preparing the compositionsinclude the step of bringing the active ingredient into association witha carrier containing one or more accessory ingredients. Thepharmaceutical compositions are typically prepared by uniformly andintimately bringing the active ingredient into association with a liquidcarrier or a finely divided solid carrier or both, and then, ifnecessary, shaping the product into the desired formulation. Thecompositions can be conveniently prepared and/or packaged in unit dosageform.

Pharmaceutical compositions containing compounds of the invention can beformulated for oral use. Suitable compositions for oral administrationinclude, but are not limited to, tablets, troches, lozenges, aqueous oroily suspensions, dispersible powders or granules, emulsions, hard orsoft capsules, syrups, elixirs, solutions, buccal patches, oral gels,chewing gums, chewable tablets, effervescent powders, and effervescenttablets. Compositions for oral administration can be formulatedaccording to any method known to those of skill in the art. Suchcompositions can contain one or more agents selected from sweeteningagents, flavoring agents, coloring agents, antioxidants, and preservingagents in order to provide pharmaceutically elegant and palatablepreparations.

Tablets generally contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients, including: inertdiluents, such as cellulose, silicon dioxide, aluminum oxide, calciumcarbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose,calcium phosphate, and sodium phosphate; granulating and disintegratingagents, such as corn starch and alginic acid; binding agents, such aspolyvinylpyrrolidone (PVP), cellulose, polyethylene glycol (PEG),starch, gelatin, and acacia; and lubricating agents such as magnesiumstearate, stearic acid, and talc. The tablets can be uncoated or coated,enterically or otherwise, by known techniques to delay disintegrationand absorption in the gastrointestinal tract and thereby provide asustained action over a longer period. For example, a time delaymaterial such as glyceryl monostearate or glyceryl distearate can beemployed. Tablets can also be coated with a semi-permeable membrane andoptional polymeric osmogents according to known techniques to formosmotic pump compositions for controlled release.

Compositions for oral administration can be formulated as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent (such as calcium carbonate, calcium phosphate, or kaolin), or assoft gelatin capsules wherein the active ingredient is mixed with wateror an oil medium (such as peanut oil, liquid paraffin, or olive oil).

Rgp inhibitors can also be administered topically as a solution,ointment, cream, gel, or suspension, as well as in mouth washes,eye-drops, and the like. Still further, transdermal delivery of Rgpinhibitors can be accomplished by means of iontophoretic patches and thelike.

Pharmaceutical compositions containing Rgp inhibitors can also be in theform of a sterile injectable aqueous or oleaginous solutions andsuspensions. Sterile injectable preparations can be formulated usingnon-toxic parenterally-acceptable vehicles including water, Ringer'ssolution, and isotonic sodium chloride solution, and acceptable solventssuch as 1,3-butane diol. In addition, sterile, fixed oils can be used asa solvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic monoglycerides, diglycerides, ortriglycerides.

In some embodiments, an Rgp inhibitor can be formulated with a polymersuch as Pluronic F127 and delivered subcutaneously. Pluronic is ahydrogel that solidifies at body temperature and can provide extendeddrug delivery over periods of time lasting from days to weeks.

Aqueous suspensions can contain one or more Rgp inhibitors in admixturewith excipients including, but not limited to: suspending agents such assodium carboxymethylcellulose, methylcellulose,oleagino-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone,gum tragacanth and gum acacia; dispersing or wetting agents such aslecithin, polyoxyethylene stearate, and polyethylene sorbitanmonooleate; and preservatives such as ethyl, n-propyl, andp-hydroxybenzoate. Dispersible powders and granules (suitable forpreparation of an aqueous suspension by the addition of water) cancontain one or more Rgp inhibitors in admixture with a dispersing agent,wetting agent, suspending agent, or combinations thereof. Oilysuspensions can be formulated by suspending an Rgp inhibitor in avegetable oil (e.g., arachis oil, olive oil, sesame oil or coconut oil),or in a mineral oil (e.g., liquid paraffin). Oily suspensions cancontain one or more thickening agents, for example beeswax, hardparaffin, or cetyl alcohol. These compositions can be preserved by theaddition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions of the invention can also be in the formof oil-in-water emulsions. The oily phase can be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, such as gum acacia or gum tragacanth;naturally-occurring phospholipids, such as soy lecithin; esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan monooleate; and condensation products of said partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate.

The use of hybrid molecules to promote active transport or nanoparticlescan be used in certain embodiments to increase blood brain barriertransport. For example liposomes, proteins, engineered peptide compoundsor antibodies that bind to the receptors that transport proteins acrossthe blood brain barrier including LPR-1 receptor, transferrin receptor,EGF-like growth factor or glutathione transporter can be used toincrease penetration into the brain. Physical techniques includingosmotic opening, ultrasound, lasers, sphenopalantine ganglionstimulation, direct intracranial, intrathecal, or intraventriculardelivery via a pump can be used.

Pharmaceutical compositions according to the invention can also includeone or more additional active agents useful in the treatment ofconditions associated with P. gingivalis infection. In certainembodiments, the invention provides a pharmaceutical compositioncomprising one or more Rgp inhibitors as described herein in combinationwith one or more additional active agents for treatment of Alzheimer'sdisease. Several therapeutics are in development and in clinical use fortreatment of Alzheimer's disease. Therapeutic strategies includelowering circulating levels of β-amyloid and tau (as described in moredetail below), stabilizing microtubules, removing atheroscleroticplaques, modulating autophagy, modulating neurotransmitter levels, andinhibiting GABA(A) α5 receptors. Such therapeutics can maintain and/orrestore cognitive function in subjects with Alzheimer's disease; slowthe decline of cognitive function; and promote neuroplasticity andrecovery of the brain.

Active agents that can be combined with Rgp inhibitors in pharmaceuticalcompositions include, but are not limited to, antibiotics (i.e.,bacteriocidal compounds and bacteriostatic compounds), cholinesteraseinhibitors, alpha-7 nicotinic receptor modulators, serotonin modulators,NMDA modulators, Aβ-targeted therapies, ApoE-targeted therapies,microglia-targeted therapies, blood/brain barrier-targeted therapies,tau-targeted therapies, complement-targeted therapies, andanti-inflammatories.

Any suitable antibiotic can be combined with one or more Rgp inhibitorsin the pharmaceutical compositions of the invention. In certainembodiments, the invention provides a pharmaceutical compositioncontaining one more Rgp inhibitors and an antibiotic having a P.gingivalis MIC₅₀ of less than 25 μg/ml. For example, the P. gingivalisMIC₅₀ of the antibiotic can be less than 20 μg/ml, less than 15 μg/ml,less than 10 μg/ml, less than 8 μg/ml, less than 6 μg/ml, or less than 5μg/ml. In some embodiments, the P. gingivalis MIC₅₀ of the antibiotic isless than 1 μg/ml. In some embodiments, the P. gingivalis MIC so of theantibiotic is less than 0.2 μg/ml.

Examples of bacteriocidal and bacteriostatic compounds include, but arenot limited to: quinolones (e.g., moxifloxacin, gemifloxacin,ciprofloxacin, oflaxacin, trovafloxacin, sitafloxacin, and the like),β-lactams (e.g., penicillins such as amoxicillin,amoxacilin-clavulanate, piperacillin-tazobactam, penicillin G, and thelike; and cephalosporins such as ceftriaxone and the like), macrolides(e.g., erythromycin, azithromycin, clarithromycin, and the like),carbapenems (e.g., doripenem, imipenem, meropinem, ertapenem, and thelike), thiazolides (e.g., tizoxanidine, nitazoxanidine, RM 4807, RM4809, and the like), tetracyclines (e.g., tetracycline, minocycline,doxycycline, eravacycline, and the like), clindamycin, metronidazole,and satranidazole. Bacteriocidal and bacteriostatic compounds alsoinclude agents that inhibit or otherwise interfere with formation ofbiofilms by anaerobic, gram-negative bacteria; such agents includeoxantel, morantel, thiabendazole, and the like. Compositions of theinvention can contain one or more Rgp inhibitors with one or more (e.g.,two, three, four, five, six, or more) bacteriocidal/bacteriostaticcompounds. Compositions containing bacteriocidal/bacteriostaticcompounds can further contain a chlorhexidine (e.g., chlorhexidinedigluconate) alone or in combination with a zinc compound (e.g., zincacetate), can also be used in combination with the administeredantibiotics.

In some embodiments, a combination of a penicillin (e.g., amoxicillin)and metronidazole or a combination of penicillin (e.g., amoxicillin),metronidazole and a tetracycline is used. In some embodiments, theantibiotic is selected from minocycline, doxycycline, metronidazole,amoxicillin, clindamycin, augmentin, satranidazole, and combinationsthereof.

Examples of suitable cholinesterase inhibitors include, but are notlimited to, donepezil, donepezil/memantine, galantamine, rivastigmine,and tacrine, as well as pharmaceutically acceptable salts thereof.Examples of suitable serotonin modulators include, but are not limitedto, idalopirdine, RVT-101, citalopram, escitalopram, fluoxetine,fluvoxamine, paroxetine, and sertraline, as well as pharmaceuticallyacceptable salts thereof. Examples of suitable alpha-7 nicotinicreceptor modulators include, but are not limited to, alpha-7 agonistssuch as encenicline and APN1125. Suitable NMD A modulators include, butare not limited to, NMD A receptor antagonists such as memantine andderivatives thereof.

Pharmaceutical compositions of the invention can also contain activeagents that are directed to biomolecular targets associated withneurological diseases. Such targets include beta amyloid peptides (alsoreferred to as beta amyloid, abeta, or Aβ), apolipoprotein E (alsoreferred to as ApoE), and microtubule-associated tau (also referred toas tau proteins, or simply as tau).

Aβ-targeted therapies include inhibitors of Aβ production (such asbeta-secretase inhibitors, gamma-secretase inhibitors, alpha-secretaseactivators), inhibitors of Aβ aggregation, inhibitors of Aβoligomerization, and up-regulators of Aβ clearance, among others (see,e.g., Jia, et al. BioMed Research International 2014. Article ID 837157,doi: 10.1155/2014/837157). Examples of Aβ-targeted therapies include butare not limited to, antibodies, pioglitazone, begacestat, atorvastatin,simvastatin, etazolate, and tramiprosate, as well as pharmaceuticallyacceptable salts thereof.

Examples of ApoE-targeted therapies include, but are not limited toretinoid X receptor agonists (see, Cramer, et al., Science 2012.335(6075): 1503-1506) and others described by Liu et al. (Nat RevNeurol. 2013. 9(2): 106-118). Tau-targeted therapies include, but arenot limited to, methylthioninium, leuco-methylthioninium, antibodies andthose described by Lee, et al. (Cold Spring Hard Perspect Med 2011; 1:a006437).

Pharmaceutical compositions of the invention can also containcomplement-targeted therapies. Such therapies target components of thecomplement system involved in the innate immune response. Complementtargeted therapies include, but are not limited to, those described byRicklin and Lambris (Nat. Biotechnology 2007. 25(11): 1265-1275).

Examples of suitable anti-inflammatories include, but are not limitedto, NSAIDs such as apazone, diclofenac, ibuprofen, indomethacin,ketoprofen, nabumetone, naproxen, piroxicam, and sulindac, as well aspharmaceutically acceptable salts thereof.

V. Methods for Inhibiting Gingipains and Treating Conditions Associatedwith P. gingivalis Infection

In another embodiment, the invention provides a method of inhibiting agingipain. The method includes contacting the gingipain with aneffective amount of a compound as described herein. In certainembodiments, the gingipain is an arginine gingipain (e.g., RgpA, RgpB,or a variant containing one or more amino acid substitutions, deletions,and/or other peptide sequence variations). Inhibiting the gingipaingenerally includes contacting the gingipain with an amount of thecompound sufficient to reduce the activity of the gingipain as comparedto the gingipain activity in the absence of the compound. For example,contacting the gingipain with the gingipain inhibitor can result in fromabout 1% to about 99% gingipain inhibition (i.e., the activity of theinhibited gingipain ranges from 99% to 1% of the gingipain activity inthe absence of the compound). The level of gingipain inhibition canrange from about 1% to about 10%, or from about 10% to about 20%, orfrom about 20% to about 30%, or from about 30% to about 40%, or fromabout 40% to about 50%, or from about 50% to about 60%, or from about60% to about 70%, or from about 70% to about 80%, or from about 80% toabout 90%, or from about 90% to about 99%. The level of gingipaininhibition can range from about 5% to about 95%, or from about 10% toabout 90%, or from about 20% to about 80%, or from about 30% to about70%, or from about 40% to about 60%. In some embodiments, contacting thegingipain with a compound as described herein will result in complete(i.e., 100%) gingipain inhibition.

As described above, infection with P. gingivalis and gingipain activityhave been linked to the development of periodontal disease, Alzheimer'sand other brain disorders, cardiovascular disease, diabetes, cancer,liver disease, kidney disease, preterm birth, arthritis, pneumonia andother disorders. See: Bostanci, et al. FEMS Microbiol Lett, 2012.333(1): 1-9; Ghizoni, et al. J Appl Oral Sci, 2012. 20(1): 104-12; Gatz,et al. Alzheimers Dement, 2006. 2(2): 110-7; Stein, et al. J Am DentAssoc, 2007. 138(10): 1314-22; quiz 1381-2; Noble, et al. J NeurolNeurosurg Psychiatry, 2009. 80(11): 1206-11; Sparks Stein, et al.Alzheimers Dement, 2012. 8(3): 196-203; Velsko, et al. PLoS ONE, 2014.9(5): e97811; Demmer, et al. J Dent Res, 2015. 94(9S): 201-S-11S;Atanasova and Yilmaz. Molecular Oral Microbiology, 2014. 29(2): 55-66;Yoneda, et al. BMC Gastroenterol, 2012. 12: 16.

Extracellular proteases produced by P. gingivalis, including ArginineGingipain A (RgpA), Arginine Gingipain B (RgpB), and Lysine Gingipain(Kgp), can also degrade a broad range of proteins in connective tissueand plasma (e.g., collagen, immunoglobulins, and proteinase inhibitors,etc.). Gingipains can enter systemic circulation and/or synoviocytes andchondrocytes, and they can also cause disruption to the kallikrein-kinincascade, blood coagulation, and host defense systems. Patients withgingipains in their joints and circulatory system may be subject togingipain-induced death of synovial cells and/or chondrocytes,contributing to osteoarthritis.

It has recently been discovered that RgpB and Kgp can infiltrate humanand dog joints, contributing to the development of osteoarthritis. It isbelieved that P. gingivalis and gingipains can infiltrate joint tissuesvia a number of routes. Gingipains can be secreted, transported to outermembrane surfaces of P. gingivalis, or released in outer membranevesicles by the bacterium. P. gingivalis has previously been identifiedin periodontal tissues, coronary arteries, aorta, and recently, theliver-release of P. gingivalis and/or gingipains from any of theseniches into the systemic circulation could result in translocation of P.gingivalis and/or gingipains to the joints. See; Travis, et al. Adv ExpMed Biol, 2000. 477: 455-65; Byrne, et al. Oral Microbiol Immunol, 2009.24(6): 469-77; Mahendra, et al. J Maxillofac Oral Surg, 2009. 8(2):108-13; Stelzel. Periodontol, 2002. 73(8): 868-70; Ishikawa, et al.Biochim Biophys Acta, 2013. 1832(12): 2035-2043.

P. gingivalis and/or gingipains may also enter joints by degrading theendothelial cells protecting the blood/joint barrier, or by a traumaticevent to the joint, such as a meniscus injury, which permanently ortransiently reduces the integrity of the joint tissues. Such adisruption in traumatic joint injury for example, may contribute to theinfiltration of P. gingivalis and/or gingipains in infected individualsand subsequent development of chronic osteoarthritis. People who are ata high risk of traumatic joint injury, including athletes in contactsports like football, could be preventatively treated with gingipaininhibitors to reduce the risk of trauma-related osteoarthritis.

P. gingivalis and gingipains may also reach the joint through othermechanisms including active transport, passive transport or macrophagedelivery. Osteoarthritis resulting from any of these mechanisms can belimited to a single joint or present in multiple joints.

Similar to humans, P. gingivalis infection and periodontal disease isone of the most common infectious diseases affecting adult dogs andcats. Dogs and cats with P. gingivalis infection and gingipains in theirjoints and circulatory system may experience periodontal disease andosteoarthritis due to gingipain-induced cell death, which could betreated or prevented according to the methods of the invention. Ageddogs spontaneously develop many features of osteoarthritis, including acommon inflammatory knee arthritis associated with degeneration of theanterior cruciate ligament (ACL). A study by Muir et al. of dogs withinflammatory knee arthritis and ACL degeneration detected DNA from arange of bacterial species in 37% of knee joints from affected dogs.Muir et al. hypothesized that bacteria may be an important causativefactor in the pathogenesis of inflammatory arthritis in dogs. In theMuir et al. study, DNA from P. gingivalis was not detected in the dogjoints. See, Muir, et al. Microb Pathog, 2007. 42(2-3): 47-55. However,similar to humans, P. gingivalis is a common oral pathogen affectingadult dogs, and could potentially translocate from the oral cavity tojoint tissues as a result of bacteremia. P. gingivalis has beendemonstrated to infect chondrocytes in vitro causing chondrocyteapoptosis, indicating a pathway for cartilage loss in osteoarthritis ofboth dogs and humans. See; Rohner, et al. Calcif Tissue Int, 2010.87(4): p. 333-40; Houle, et al. FEMS Microbiol Lett, 2003. 221(2): p.181-5; Kataoka, et al. FASEB J, 2014. 28: 3564-3578; Pischon, et al. AnnRheum Dis, 2009. 68(12): p. 1902-7.

Rgp inhibitors can therefore be used to treat diseases and conditions,such as brain disorders, caused by or otherwise affected by P.gingivalis. Accordingly, another aspect of the invention provides amethod of treating a disease or condition associated with P. gingivalisinfection. The method includes administering an effective amount of acompound or a composition of the invention, as described above, to asubject in need thereof.

In certain embodiments, compounds of the invention inhibit active Rgp inthe brain of a mammal, e.g., a human or an animal (e.g., a dog), and arecytoprotective or neuroprotective. By “neuroprotective,” it is meantthat the compounds prevent aberrant changes to neurons or death ofneurons. Compounds of the invention are therefore useful, e.g., intreatment of a brain disorder (e.g., a neurodegenerative disease (e.g.,Alzheimer's disease, Down's syndrome, epilepsy, autism, Parkinson'sdisease, essential tremor, fronto-temporal dementia, progressivesupranuclear palsy, amyotrophic lateral sclerosis, Huntington's disease,multiple sclerosis, mild cognitive impairment, age associated memoryimpairment, chronic traumatic encephalopathy, stroke, cerebrovasculardisease, Lewy Body disease, multiple system atrophy, schizophrenia anddepression, etc.), diabetes, cardiovascular disease, arthritis,rheumatoid arthritis, osteoarthritis, infectious arthritis, psoriaticarthritis, retinal disorders (e.g., age related macular degeneration)and glaucoma.

In some embodiments, the disease or condition is selected from a braindisorder, periodontal disease, diabetes, a cardiovascular disease,arthritis, rheumatoid arthritis, osteoarthritis, preterm birth,pneumonia, cancer, a kidney disease, a liver disease, a retinaldisorder, and glaucoma.

In some embodiments, the disease or condition is a brain disorder.

In some embodiments, the brain disorder is selected from Alzheimer'sdisease, Down's syndrome, epilepsy, autism, Parkinson's disease,essential tremor, fronto-temporal dementia, progressive supranuclearpalsy, amyotrophic lateral sclerosis, Huntington's disease, multiplesclerosis, mild cognitive impairment, age associated memory impairment,chronic traumatic encephalopathy, stroke, cerebrovascular disease, LewyBody disease, multiple system atrophy, schizophrenia, and depression.

In some embodiments, the brain disorder is Alzheimer's disease.

In some embodiments, the method further includes administering to thesubject one or more active agents selected from a cholinesteraseinhibitor, a serotonin modulator, an NMDA modulator, an Aβ targetedtherapy, an ApoE targeted therapy, a microglia targeted therapy, a bloodbrain barrier targeted therapy, a tau targeted therapy, a complementtargeted therapy, and an anti-inflammatory.

In some embodiments, the disease or condition is periodontal disease. Insome embodiments, the disease or condition is a liver disease. In someembodiments, the liver disease is non-alcoholic steatohepatitis. In someembodiments, the disease or condition is a retinal disorder. In someembodiments, the retinal disorder is age-related macular degeneration.

In some embodiments, the disease or condition is cancer. In someembodiments, the cancer is breast cancer, oral cancer, pancreaticcancer, or glioblastoma multiforme.

Rgp inhibitors as described herein can be administered at any suitabledose in the methods of the invention. In general, an Rgp inhibitor isadministered at a dose ranging from about 0.1 milligrams to about 1000milligrams per kilogram of a subject's body weight (i.e., about 0.1-1000mg/kg). The dose of Rgp inhibitor can be, for example, about 0.1-1000mg/kg, or about 1-500 mg/kg, or about 25-250 mg/kg, or about 50-100mg/kg. The dose of Rgp inhibitor can be about 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85, 90, 95, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950 or 1000 mg/kg. The dosages can be varied depending upon therequirements of the patient, the severity of the disorder being treated,and the particular formulation being administered. The dose administeredto a patient should be sufficient to result in a beneficial therapeuticresponse in the patient. The size of the dose will also be determined bythe existence, nature, and extent of any adverse side-effects thataccompany the administration of the drug in a particular patient.Determination of the proper dosage for a particular situation is withinthe skill of the typical practitioner. The total dosage can be dividedand administered in portions over a period of time suitable to treat tothe disease or condition.

Rgp inhibitors can be administered for periods of time which will varydepending upon the nature of the particular disorder, its severity, andthe overall condition of the subject to whom the Rgp inhibitor isadministered. Administration can be conducted, for example, hourly,every 2 hours, three hours, four hours, six hours, eight hours, or twicedaily including every 12 hours, or any intervening interval thereof.Administration can be conducted once daily, or once every 36 hours or 48hours, or once every month or several months. Following treatment, asubject can be monitored for changes in his or her condition and foralleviation of the symptoms of the disorder. The dosage of the Rgpinhibitor can either be increased in the event the subject does notrespond significantly to a particular dosage level, or the dose can bedecreased if an alleviation of the symptoms of the disorder is observed,or if the disorder has been remedied, or if unacceptable side effectsare seen with a particular dosage.

A therapeutically effective amount of an Rgp inhibitor can beadministered to the subject in a treatment regimen comprising intervalsof at least 1 hour, or 6 hours, or 12 hours, or 24 hours, or 36 hours,or 48 hours between dosages. Administration can be conducted atintervals of at least 72, 96, 120, 144, 168, 192, 216, or 240 hours(i.e., 3, 4, 5, 6, 7, 8, 9, or 10 days). In certain embodiments,administration of one or more Rgp inhibitors is conducted in a chronicfashion over periods ranging from several months to several years.Accordingly, some embodiments of the invention provide a method oftreating a disease or condition associated with P. gingivalis infectionas described above, wherein the compound is administered to the subjectfor at least one year. In some embodiments, the compound is administeredto the subject for at least 10 years. In some embodiments, the compoundis administered to the subject for at least 60 years.

Administration of Rgp inhibitors according to the methods of theinvention typically results in the reduction of circulating levels ofactive Rgp in a subject and/or the reduction of active Rgp in the brain.In certain embodiments, administration of an Rgp inhibitor according tothe methods of the invention results in at least a 20% reduction ofcirculating levels of active Rgp and/or at least a 20% reduction ofactive Rgp in the brain. For example, the circulating levels of Rgpand/or the levels of Rgp in the brain are preferably reduced by fromabout 25% to about 95%, or from about 35% to about 95%, or from about40% to about 85%, or from about 40% to about 80% as compared to thecorresponding levels of Rgp 24 hours prior to the first administrationof the Rgp inhibitor.

Rgp inhibitors can be administered alone or in combination with one ormore additional therapeutically active agents, as described above. Theone or more additional therapeutically effective agents include, e.g.:(i) a pharmaceutically acceptable agent which inhibits Rgp A, RgpB,and/or Kgp production, translocation of RgpA, RgpB, and/or Kgp intosystemic circulation or brain, and/or pathological (e.g., neurotoxiceffects) of RgpA, RgpB, and/or Kgp in a mammal; (ii) an antibacterialagent which is bacteriostatic or bacteriocidal with respect to P.gingivalis; (iii) one or more antibodies which bind to RgpA, RgpB and/orKgp (e.g., 18E6, which binds to the first half of the immunoglobulindomain of RgpB; Kgp-specific monoclonal antibody, 7B9, which recognizesan epitope within the Kgp catalytic domain; the RgpA antibody 61Bg 1.3,humanized versions of any of the foregoing, etc.); (iv) epitopes ofantibodies which bind to RgpA, RgpB and/or Kgp or other proteinsexpressed by P. gingivalis; and (v) combinations of any of theforegoing.

The additional therapeutically active agents also include Aβ peptideslevel reducers, pathogenic level tau reducers, microtubule stabilizers,agents capable or removing atherosclerotic plaques, agents that lowercirculating levels of β-amyloid and tau, modulators of autophagy,neurotransmitter level regulators, GABA(A) α5 receptors inhibitors, andadditional agents that help maintain and/or restore cognitive functionand functional deficits of Alzheimer's disease, and/or slow down declinein cognitive functions and functional deficits in Alzheimer's disease.

Pharmaceutical compositions of the invention can contain one or more Rgpinhibitors as described herein in combination with ritonavir (RTV),which can increase bioavailability and increase blood brain barrierpenetration. For example, ritonavir is commonly combined with oralpeptidic HIV protease inhibitors to increase plasma levels by inhibitingthe P450 3 A4 enzyme and thus decreasing first-pass metabolism (see,Walmsley, et al., N Engl J Med, 2002. 346(26): 2039-46). In addition,RTV binds to P-glycoprotein, a transmembrane efflux pump that is foundin many tissues, including the blood brain barrier, allowingco-administered compounds better access to the brain (see, Marzolini, etal., Mol Pharm, 2013. 10(6): 2340-9). Therefore, a combination of RTVand Rgp inhibitors can be used to increase plasma concentrations andbrain levels of the gingipain inhibitors. As described in U.S. patentapplication Ser. No. 14/875,416, oral administration of RTV 15 minutesprior to the Kgp inhibitor Kyt-36 increases the half-life therefore itis expected that RTV will also increase the half-life of other gingipaininhibitors.

In some embodiments, compounds of the invention can be administered withnatural gingipain inhibitors including melabaricone C, isolated fromnutmeg or polyphenolic compounds derived from plants, such as cranberry,green tea, apple, and hops can be administered in conjunction fortreatment or prevention of brain disorders. Naturally and unnaturallyoccurring antimicrobial peptides including: κ-casein peptide (109-137)34, histatin 5, and CL(14-25), CL(K25A) and CL(R24A, K25A), can also beadministered in conjunction with the Rgp inhibitors of the invention,(see, e.g., Taniguchi et al., Biopolymers, 2014. 102(5): 379-89).

Rgp inhibitors as described herein can be administered with antibodiestargeting gingipains or other P. gingivalis proteins. Antibodies mayrely on damage to the blood brain barrier for access to the brain orperipheral interference with gingipains and P. gingivalis propagation.Antibodies can also help to stimulate the efficacy of the immune systemin clearing the bacteria. New or existing antibodies to RgpA, RgpB, orKgp can be utilized including 18E6 and 7B9. An RgpA antibody 61BG 1.3has previously demonstrated efficacy topically in prevention ofrecolonization by P. gingivalis after periodontal treatment. See, Boothet al., Infect Immun, 1996. 64(2): 422-7. Antibodies would preferably behumanized for use in humans. Methods known to those in the field fordelivery of biologics to improve half-life and brain penetration can beused including, but not limited to, intravenous delivery, subcutaneousdelivery, intranasal delivery, intrathecal delivery, intra-articulardelivery, vector transport, and direct brain delivery.

The methods of the invention also encompass administration of Rgpinhibitors as described herein with one or more of the followingadditional therapeutically active agents or pharmaceutically acceptablesalts thereof: an arginine derivative; histatin 5; baculovirus p35; asingle point mutant of cowpox viral cytokine-response modifier (CrmA(Asp>Lys)); phenylalanyl-ureido-citrullinyl-valyl-cycloarginal(FA-70C1); (acycloxy)methyl ketone (Cbz-Phe-Lys-CH₂OCO-2,4,6-Me₃Ph);peptidyl chloro-methyl ketones (e.g., chloromethyl ketone derivatives ofarginine, chloromethyl ketone derivatives of lysine, and the like);fluoro-methyl ketones; bromo-methyl ketones; ketopeptides;1-(3-phenylpropionyl)piperidine-3(R,S)-carboxylic acid[4-amino-1(S)-(benzothiazole-2-carbonyl)butyl]amide (A71561); azapeptidefumaramide; aza-peptide Michael acceptors; benzamidine compounds;acyclomethylketone; activated factor X inhibitors (e.g., DX-9065a);cranberry nondialyzable fraction; cranberry polyphenol fraction;pancreatic trypsin inhibitor; Cbz-Phe-Lys-CH₂O—CO-2,4,6-Me₃-Ph; E-64;chlorhexidine; zinc (e.g., zinc acetate); or a combination of two, threeor more of any of foregoing. In some of these embodiments, Zn canenhance potency and selectivity of the compounds (e.g., chlorhexidine,benzamidine, etc.) used in the methods of the invention.

An Rgp inhibitor of the invention can be administered in the samecomposition as an additional therapeutically active agent.Alternatively, the additional therapeutically active agent can beadministered separately before, concurrently with, or afteradministration of the Rgp inhibitor.

VI. Examples Example 1. Preparation ofN-(1-aminoisoquinolin-7-yl)-N-methylcyanamide (1)

To a solution of compound 1.5 (3 g, 12.4 mmol, 1 eq) in NMP (30 mL) wasadded NH₃.H₂O (30 mL). The mixture was stirred 150° C. for 15 hours. Thereaction mixture was quenched by addition H₂O 100 mL at 25° C., and thenextracted with EtOAC (100 mL×3). The combined organic layers were washedwith saturated brines (15 mL×1), dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a crudeproduct. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1:1). Compound 1.4 (2.0 g, 9 mmol, 72.42%yield) was obtained as a brown oil. LCMS (ESI): m/z: [M+H] calcd forC₉H₇N₂Br:223; found 223; RT=1.049 min.

To a solution of compound 1.4 (1.90 g, 8.52 mmol, 1 eq) in THF (40 mL)was added TEA (3.45 g, 34.08 mmol, 4.73 mL, 4 eq) and Boc₂O (4.65 g,21.30 mmol, 4.89 mL, 2.50 eq) and DMAP (312.18 mg, 2.56 mmol, 0.30 eq).The mixture was stirred at 25° C. for 15 hours. The reaction mixture wasquenched by addition H₂O 50 mL at 25° C. and extracted with EtOAc (50mL×3). The combined organic layers were washed with saturated brines (20mL×1), dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure to give a crude product. The residue was purifiedby column chromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1).Compound 1.3 (2.50 g, 5.91 mmol, 69.32% yield) was obtained as a yellowoil. ¹H NMR (400 MHz, methanol-d₄) 5 ppm 1.16-1.49 (m, 21H) 7.89 (d,J=5.77 Hz, 1H) 7.92-8.03 (m, 2H) 8.10 (s, 1H) 8.42 (d, J=5.77 Hz, 1H).LCMS (ESI): m/z: [M+H] calcd for C19H23N2BrO4:423; found 423; RT=0.957min.

To a solution of compound 1.3 (1.50 g, 3.54 mmol, 1 eq) in DMF (30 mL)was added tert-butyl N-methylcarbamate (557.21 mg, 4.25 mmol, 1.20 eq),Cs₂CO₃ (3.46 g, 10.62 mmol, 3 eq), 4, 5-BIS (DIPHENYLPHOSPHINO)-9,9-DIMETHYLXANTHENE (819.33 mg, 1.42 mmol, 0.40 eq) and Pd₂(dba)₃ (972.49mg, 1.06 mmol, 0.30 eq). The mixture was stirred at 80° C. for 2 hours.The reaction mixture was quenched by addition H₂O 50 mL at 25° C. andextracted with ethyl acetate (50 mL×3). The combined organic layers werewashed with saturated brines (15 mL×1), dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give acrude product. The residue was purified by column chromatography (SiO₂,DCM:MeOH=10:1). Compound 1.2 (1.20 g, 2.53 mmol, 71.58% yield) wasobtained as a yellow oil. LCMS (ESI): m/z: [M+H] calcd forC25H35N3O6:474; found 474; RT=0.936 min.

A mixture of compound 1.2 (1.20 g, 2.53 mmol, 1 eq) in HCl/EtOAc (10 mL)was stirred at 25° C. for 15 hours. The mixture was concentrated underreduced pressure to give compound 1.1 (600 mg, 2.12 mmol, 83.92% yield,3HCl) was obtained as a yellow solid. ¹H NMR (400 MHz, DEUTERIUM OXIDE)5 ppm 7.08 (d, J=7.06 Hz, 1H) 7.38 (d, J=6.84 Hz, 1 H) 7.59-7.69 (m, 2H)7.78 (d, J=8.60 Hz, 1H). LCMS (ESI): m/z: [M+H] calcd forC₁₀H₁₁N₃O₆:174; found 174; RT=0.236 min.

To a solution of compound 1.1 (100 mg, 406.27 μmol, 1 eq, 2HCl) in DMSO(3 mL) was added NaHCO₃ (170.65 mg, 2.03 mmol, 79.01 μL, 5 eq) and BrCN(43.03 mg, 406.27 μmol, 29.88 μL, 1 eq). The mixture was stirred at 60°C. for 0.5 hour. The residue was purified by prep-HPLC (neutralcondition). Product 1 (2 mg, 8.07 μmol, 1.99% yield, 80% purity) wasobtained as a yellow solid. ¹H NMR (400 MHz, DMSO-dis) 8 ppm 3.43-3.48(m, 3H) 6.78-6.84 (m, 2H) 6.88-6.96 (m, 1H) 6.89-6.92 (m, 1H) 7.51-7.55(m, 1H) 7.71 (d, J=2.43 Hz, 1H) 7.75 (d, J=5.73 Hz, 1H) 7.79 (d, J=8.82Hz, 1H). LCMS (ESI): m/z: [M+H] calcd for C11H10N4:199; found 199;RT=2.246 min.

Example 2. Preparation of3-((2-aminopyridin-4-yl)oxy)pyrrolidine-1-carbonitrile (2)

To a solution of compound 2.3 (800 mg, 7.27 mmol, 1 eq) in DCM (30 mL)was added compound 50 (1.61 g, 7.27 mmol, 1 eq), PPh₃ (2.86 g, 10.91mmol, 1.50 eq) and DIAD (2.21 g, 10.91 mmol, 2.12 mL, 1.50 eq) in turnat 0° C. under N₂. The resulting mixture was stirred at 25° C. for 16hours. The reaction mixture was added water 20 mL, extracted with EtOAc(20 mL×3). The combined organic layers were washed with brine (20 mL×1),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1:1). Compound 2.2 (750 mg, 2.39 mmol,32.92% yield) was obtained as a white solid. ¹H NMR (400 MHz,chloroform-d) ppm 2.09-2.20 (m, 2H) 3.51-3.74 (m, 4H) 4.45 (s, 2H) 4.90(s, 1H) 5.93 (s, 1H) 6.21 (d, J=6.0 Hz, 1H) 7.33-7.37 (m, 5H) 7.91 (d,J=6.4 Hz, 1H).

H₂ was bubbled into a solution of compound 2.2 (400 mg, 1.28 mmol, 1 eq)and Pd/C (40 mg) in MeOH (40 mL) at 25° C. under 50 psi for 16 hours.The reaction mixture was filtered, the filter was concentrated underreduced pressure to remove solvent. Compound 2.1 (200 mg, 1.12 mmol,87.18% yield) was obtained as a yellow oil. ¹H NMR (400 MHz,methanol-d4) ppm 1.93-1.97 (m, 2H) 2.09-2.12 (m, 2H) 2.88-3.09 (m, 4H)4.90-4.93 (m, 1H) 6.06 (d, J=2.0 Hz, 1H) 6.20 (dd, J=6.0 Hz, 2.0 Hz, 1H)7.69 (d, J=6.4 Hz, 1H). LCMS (ESI): m/z: [M+H] calcd forC8H11N3O.2(C2HF302):166; found 166; RT=0.214 min.

To a solution of compound 2.1 (100 mg, 557.97 μmol, 1 eq) in THF (5 mL)was added DIEA (144.22 mg, 1.12 mmol, 194.89 μL, 2 eq) and BrCN (59.10mg, 557.97 μmol, 41.04 μL, 1 eq) in turn at 0° C. under N₂. Theresulting mixture was stirred at 0° C. for 2 hours. The reaction mixturewas added water 10 mL, extracted with EtOAc (10 mL×3). The combinedorganic layers were washed with brine (20 mL×1), dried over Na₂SO₄,filtered and concentrated under reduced pressure to give a residue. Theresidue was purified by p-TLC (SiO₂, Ethyl acetate=0:1). Product 2 (9mg, 44.07 μmol, 7.90% yield) was obtained as a white solid. LCMS (ESI):m/z: [M+H] calcd for C₁₀H₁₂N₄O:204; found 205; RT=1.672 min. ¹H NMR (400MHz, chloroform-d) ppm 2.12-2.26 (m, 2H) 3.59-3.69 (m, 4H) 4.44 (s, 2H)4.92 (s, 1H) 5.92 (d, J=2.0 Hz, 1H) 6.20 (dd, J=6.4 Hz, 2.0 Hz, 1H) 7.93(d, J=6.0 Hz, 1H)

Example 3. Preparation of2-(((2-aminopyridin-4-yl)oxy)methyl)pyrrolidine-1-carbonitrile (3)

To a mixture of compound 3.5 (700 mg, 3.48 mmol, 1 eq) and4-methylbenzenesulfonyl chloride (796.15 mg, 4.18 mmol, 1.20 eq) in DCM(15 mL) was added DMAP (68.02 mg, 556.80 μmol, 0.16 eq) and TEA (528.21mg, 5.22 mmol, 723.58 μL, 1.50 eq) in one portion at 0° C. under N₂. Themixture was then heated to 25° C. and stirred for 10 hours. The reactionmixture was diluted with H₂O 20 mL and extracted with EtOAc 45 mL (15mL×3). The combined organic layers were washed with brine 20 mL (20mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give compound 3.4

(1.30 g, crude) as a colorless oil. LCMS (ESI): m/z: [M+H] calcd forC₁₇H₂₅NSO₅: 355; found 300; RT=0.866 min.

To a solution of compound 3.3 (443.30 mg, 4.03 mmol, 1.10 eq) in DMF (20mL) was added NaH (131.76 mg, 5.49 mmol, 1.50 eq) portionwise at 25° C.under N₂. The mixture was stirred at 25° C. for 30 mins, then was addedcompound 3.4 (1.30 g, 3.66 mmol, 1 eq). The mixture was heated to 60° C.and stirred for 9.5 hours. The reaction mixture was diluted with H₂O 20mL and extracted with EtOAc 45 mL (15 mL×3). The combined organic layerswere washed with brine 20 mL (20 mL×1), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, DCM:MeOH=20:1) to give compound3.2 (200 mg, 681.76 μmol, 18.63% yield) as a colorless oil. LCMS (ESI):m/z: [M+H] calcd for C₁₅H₂₃N₃O₃: 293; found 294; RT=0.663 min.

Compound 3.2 (200 mg, 681.76 μmol, 1 eq) was added into a solution ofHCl/MeOH (5 mL). The mixture was stirred at 25° C. for 15 hours. Thereaction mixture was concentrated under reduced pressure to givecompound 3.1 (220 mg, crude) as a yellow solid. LCMS (ESI): m/z: [M+H]calcd for C₁₀H₁₅N₃O: 193; found 194; RT=0.161 min.

To a mixture of compound 3.1 (100 mg, 517.46 μmol, 1 eq) and DIEA(267.51 mg, 2.07 mmol, 361.50 μL, 4 eq) in DCM (2 mL) was addedcarbononitridic bromide (54.81 mg, 517.46 μmol, 38.06 μL, 1 eq) in oneportion at 0° C. under N₂. The mixture was stirred at 0° C. for 30 mins.The reaction mixture diluted with H₂O 3 mL and extracted with DCM 9 mL(3 mL×3). The combined organic layers were washed with brine 4 mL (4mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by prep-HPLC(neutral condition) to give product 3 (40 mg, 183.28 μmol, 35.42% yield)as a yellow solid. LCMS (ESI): m/z: [M+H] calcd for C₁₁H₁₄N₄O: 218;found 219; RT=1.884 min. ¹H NMR (400 MHz, chloroform-d) δ ppm 1.83-1.93(m, 1H) 1.93-2.05 (m, 2H) 2.07-2.18 (m, 1H) 3.38-3.58 (m, 2H) 3.89-4.06(m, 3H) 4.46 (br s, 2H) 6 (d, J=2.01 Hz, 1H) 6.25 (dd, J=6.02, 2.01 Hz,1H) 7.90 (d, J=5.52 Hz, 1H).

Example 4. Preparation ofN-((4-aminofuro[3,2-c]pyridin-2-yl)methyl)-N-methylcyanamide (4)

To a solution of compound 4.7 (500 mg, 3.26 mmol, 1 eq) in PhOH (2.45 g,26.05 mmol, 2.29 mL, 8 eq) was added KOH (365.37 mg, 6.51 mmol, 2 eq) at25° C. under N₂. The resulting mixture was stirred at 140° C. for 16hrs. TLC (PE:EtOAc=5:1, Rf=0.49) showed the reaction was successful. Thereaction mixture was added water (10 mL), extracted with EtOAc (15mL×3). The organic phase was separated, washed with saturated NaCl (15mL) and dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue of compound 4.6 (0.5 g, crude) as a yellowoil which was combined with a second batch for a total of 1.5 g.

To a mixture of compound 4.6 (1.5 g, 7.10 mmol, 1 eq) in NH₄OAc (12.50g, 162.20 mmol, 22.84 eq) in one portion under N₂. The mixture wasstirred at 140° C. for 12 hours, then heated to 180° C. and stirred for12 hours. The mixture was allowed to cool to ambient temperature, afterwhich 3 N sodium hydroxide (20 ml) was added with stirring. The thusobtained solution was extracted with ethyl acetate (2×10 ml) and thecombined organic layers were extracted with 2 N hydrochloric acid (20ml). Subsequently, the pH of the aqueous layer was adjusted to 12 with 2N sodium hydroxide. Extraction with ethyl acetate (20 ml) then affordedan organic layer, which was washed with brine (10 ml), dried andconcentrated under reduced pressure to give compound 4.5 (900 mg, 6.71mmol, 94.48% yield) was obtained as light yellow solid. LCMS (ESI): m/z:[M+H] calcd for C7H6N2O:135; found 135; RT=0.199 min.

To a mixture of compound 4.5 (900 mg, 6.71 mmol, 1 eq) and benzoylbenzoate (3.04 g, 13.42 mmol, 2.53 mL, 2 eq) in Py (10 mL) in oneportion under N₂. The mixture was stirred at 150° C. for 8 hours. Themixture was poured into 500 mL of water. The resulting precipated wasfiltered off, washed with water, and dried to afford crude product ofcompound 4.4 (1.5 g, 6.30 mmol, 93.84% yield) was obtained as whitesolid. LCMS (ESI): m/z: [M+H] calcd for C14H10N2O2:239; found 239;RT=1.582 min.

To a mixture of compound 4.4 (500 mg, 2.10 mmol, 1 eq) in THF (3 mL) wasadded n-BuLi (2.5 M, 2.10 mL, 2.5 eq) in one portion at −78° C. underN2. The mixture was stirred at −78° C. for 1 h, then was added DMF(613.58 mg, 8.39 mmol, 645.87 μL, 4 eq) dropwise and stirred at −78° C.for 1 h. TLC (PE:EtOAc=3:1, Rf=0.4) showed the reaction was successful.The reaction mixture was quenched by addition H₂O (20 mL), and extractedwith EtOAc (10 mL×3). The combined organic layers were washed withsaturated brines (10 mL×2), dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a crude productof compound 4.3 (0.24 g, crude) as white solid.

To a mixture of compound 4.3 (200 mg, 751.17 μmol, 1 eq) in MeNH2(116.65 mg, 3.76 mmol, 5 eq) stirred at 25° C. for 1 h, was addedNaBH3CN (188.82 mg, 3 mmol, 4 eq) then was added AcOH (67.66 mg, 1.13mmol, 64.44 μL, 1.5 eq) until pH=0.3. The mixture was stirred at 25° C.for 11 hr. TLC (PE:EtOAc=1:1, Rf=0.3) showed the reaction wassuccessful. The reaction residue was diluted with H2O (20 mL) andextracted with EtOAc (20 mL×3). The aqueous layer added 5N aq.NaOH untilPH=11 and extracted with EtOAc (20 mL×4). The combined organic layerswere washed with brine 20 mL (10 mL×2), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue of compound 4.2(100 mg, crude) was obtained as white solid.

To a mixture of compound 4.2 (100 mg, 355.48 μmol, 1 eq) in dioxane (0.5mL) was added HCl (0.5 mL) in one portion at 80° C. under N₂. Themixture was stirred at 80° C. for 2 hours. The reaction solution wasconcentrated in vacuum to obtain compound 4.1 (100 mg, crude) as blackbrown solid. LCMS (ESI): m/z: [M+H] calcd for C9H11N3O:178; found: 178;RT=0.162 min.

To a mixture of compound 4.1 (100 mg, 399.80 μmol, 1 eq, 2HCl) in DMF (2mL) was added DIPEA (206.69 mg, 1.60 mmol, 278.55 μL, 4 eq) in oneportion at 25° C. under N₂. Then BrCN (46.58 mg, 439.78 μmol, 32.35 μL,1.1 eq) added at 0° C. and stirred at 0° C. for 20 min. The residue waspurified by prep-HPLC (neutral condition) to give product 4 (1.95 mg,9.64 μmol, 2.41% yield) was obtained as light yellow oil. ¹H NMR (400MHz, methanol-d₄) δ ppm 7.76 (d, J=6.11 Hz, 1H) 6.97 (s, 1H) 6.83 (dd,J=6.11, 0.85 Hz, 1H) 4.36 (s, 2H) 2.92 (s, 3H). LCMS (ESI): m/z: [M+H]calcd for C10H10N4O:203; found 203; RT=1.683 min.

Example 5. Preparation ofN-(2-(2-aminopyridin-4-yl)ethyl)-N-isopropylcyanamide (5)

To a solution of compound 5.9 (20 g, 115.60 mmol, 1 eq) in THF (400 mL)was added Boc₂O (63.07 g, 289 mmol, 66.39 mL, 2.50 eq), DMAP (4.24 g,34.68 mmol, 0.30 eq) and TEA (35.09 g, 346.80 mmol, 48.07 mL, 3 eq) at25° C. under N₂. The resulting mixture was stirred for 16 hours. Thereaction mixture was added water 100 mL, extracted with EtOAc (300mL×3). The organic phase was separated, washed with NH4Cl 100 mL anddried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=5:1). Compound 5.8 (27.70 g, 74.21 mmol,64.20% yield) was obtained as a colorless oil.

To a solution of compound 5.8 (27.70 g, 74.21 mmol, 1 eq) in DMF (460mL) was added TEA (15.02 g, 148.43 mmol, 20.57 mL, 2 eq), P(o-tolyl)₃(6.78 g, 22.26 mmol, 0.30 eq), Pd₂(dba)₃ (6.80 g, 7.42 mmol, 0.10 eq)and methyl prop-2-enoate 5.7 (25.56 g, 296.86 mmol, 26.62 mL, 4 eq) inturn at 25° C. under N₂. The resulting mixture was stirred at 80° C. for13 hours. The reaction mixture was added H₂O 500 mL at 25° C., extractedwith EtOAc (500 mL×3). The combined organic layers were washed withsaturated brine 200 mL (200 mL×1), dried over dry Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=50:1 to 10:1). Compound 5.6 (24 g, 63.42 mmol, 85.46% yield) wasobtained as a white solid.

To a solution of compound 5.6 (24 g, 63.42 mmol, 1 eq) in MeOH (1.50 L)was added Pd—C under Ar₂. The suspension was degassed under vacuum andpurged with H₂ several times. The mixture was stirred under H₂ (50 psi)at 25° C. for 24 hours. Pd/C was filtered off by filtration. Thefiltrate was concentrated to dry. Compound 5.5 (22 g, 57.83 mmol, 91.19%yield) was obtained as a white solid.

To a mixture of compound 5.5 (6 g, 15.77 mmol, 1 eq) in MeOH (60 mL) wasadded NaOH (1.89 g, 47.31 mmol, 3 eq) in one portion at 25° C. Themixture was stirred at 25° C. for 17 h. The pH was adjusted to around 4by progressively adding citric acid. Then the solid was precipitate out,filtered to give compound 5.4 (4 g, 10.92 mmol, 69.22% yield) as a whitesolid. LCMS (ESI): m/z: [M−H] calcd for C₁₃H₁₈N₂O₄: 266; found 265;RT=0.859 min. ¹H NMR (400 MHz, methanol-d₄) δ ppm 1.45-1.59 (m, 7H)2.56-2.70 (m, 2H) 2.83-2.98 (m, 2H) 6.91 (dd, 0.7=5.18, 1.43 Hz, 1H)7.71 (d, J=0.66 Hz, 1H) 8.06 (d, J=5.07 Hz, 1H).

To a mixture of compound 5.4 (2 g, 7.51 mmol, 1 eq) and DPPA (2.48 g,9.01 mmol, 1.95 mL, 1.20 eq) in dioxane (20 mL) was added TEA (1.52 g,15.02 mmol, 2.08 mL, 2 eq) in one portion at 25° C. under N₂. Themixture was stirred at 25° C. for 10 min, then heated to 90° C. andstirred for 2 hours. Then the mixture was added dropwise into a solutionof NaOH (1.20 g, 30.04 mmol, 4 eq) and stirred for 18 hours at 25° C.The pH was adjusted to around 6 by progressively adding 1M HCl, then themixture was partitioned between EtOAc (30) and water (30). The combinedorganic layers were washed with brine 30 mL (30 mL×2), dried overNa₂SO₄, filtered and concentrated under reduced pressure to givecompound 5.3 (800 mg, 3.37 mmol, 44.89% yield) as a white solid. LCMS(ESI): m/z: [M+H] calcd for C₁₂H₁₉N₃O₂: 237; found 294; RT=1.547 min.

To a solution of compound 5.3 (800 mg, 3.37 mmol, 1 eq) in DCM (15 mL)was added acetone (195.80 mg, 3.37 mmol, 247.85 μL, 1 eq) in one portionat 25° C. under N₂. Then the pH was adjusted to around 5 byprogressively adding glacial acetic acid. The mixture was stirred at 25°C. for 2 hour. Then the mixture was added NaBH(OAc)₃ (1.81 g, 8.56 mmol,2.54 eq) in one portion at 0° C. under N₂, then heated to 25° C. andstirred for 16 hours. The reaction was quenched by addition of 10 mL ofH₂O, a clear yellow-brown solution was obtained which was extracted byDCM (3×15 mL). The residue was purified by prep-HPLC (TFA condition) togive compound 5.2 (400 mg, 1.43 mmol, 42.43% yield) as a white solid.LCMS (ESI): m/z: [M+H] calcd for C₁₅H₂₅N₃O₂: 279; found 280; RT=1.473min. ¹H NMR (400 MHz, methanol-d₄) δ ppm 1.33 (dd, J=6.50, 1.21 Hz, 13H)1.55 (s, 9H) 2.97-3.12 (m, 5H) 3.35-3.45 (m, 3H) 6.83 (dd, J=6.73, 1.65Hz, 1H) 6.90 (dd, J=1.54, 0.66 Hz, 1H) 7.20 (dd, J=5.73, 1.54 Hz, 1H)7.55 (d, J=0.66 Hz, 1H) 7.78 (d, J=6.84 Hz, 1H) 8.19 (d, J=5.95 Hz, 1H).

Compound 5.2 (200 mg, 715.87 μmol, 1 eq) was added into a solution ofHCl/EtOAc (5 mL). The mixture was stirred at 25° C. for 14 hours. Thereaction mixture was concentrated under reduced pressure to givecompound 5.1 (180 mg, crude) as a yellow solid. LCMS (ESI): m/z: [M+H]calcd for C₁₀H₁₇N₃: 179; found 180; RT=0.250 min.

To a mixture of compound 5.1 (110 mg, 613.63 μmol, 1 eq) and DIEA(317.22 mg, 2.45 mmol, 428.68 μL, 4 eq) in THF (2 mL) was added dropwisea solution of BrCN (65 mg, 613.63 μmol, 45.14 μL, 1 eq) in THF (2 mL),and stirring at 0° C. for 10 mins. The reaction mixture was partitionedbetween EtOAc 2 mL and water 2 mL. The organic phase was separated,washed with brine 2 mL (2 mL×1), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by prep-TLC (SiO2, CH3CN:EtOAc=3:1) to give product 5 (11 mg,53.85 μmol, 8.78% yield) as a light yellow oil. LCMS (ESI): m/z: [M+H]calcd for C₁₁H₁₆N₄: 204; found 205; RT=1.164 min. ¹H NMR (400 MHz,chloroform-d) δ ppm 1.22 (d, J=6.62 Hz, 5H) 2.80-2.89 (m, 1H) 2.84 (t,J=7.28 Hz, 1H) 3.03-3.13 (m, 1H) 3.20 (t, J=7.28 Hz, 2H) 6.44 (s, 1H)6.53 (dd, J=5.51, 1.32 Hz, 1H) 7.98 (d, J=5.51 Hz, 1H).

Example 6. Preparation ofN-(3-(2-aminopyridin-4-yl)propyl)-N-isopropylcyanamide (6)

To a solution of compound 6.9 (20 g, 115.60 mmol, 1 eq) in THF (400 mL)was added Boc₂O (63.07 g, 289 mmol, 66.39 mL, 2.50 eq), DMAP (4.24 g,34.68 mmol, 0.30 eq) and TEA (35.09 g, 346.80 mmol, 48.07 mL, 3 eq) at25° C. under N₂. The resulting mixture was stirred for 16 hours. Thereaction mixture was added water 100 mL, extracted with EtOAc (300mL×3). The organic phase was separated, washed with NH4Cl 100 mL anddried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=5:1). Compound 6.8 (27.70 g, 74.21 mmol,64.20% yield) was obtained as a colorless oil.

To a solution of compound 6.8 (27.70 g, 74.21 mmol, 1 eq) in DMF (460mL) was added TEA (15.02 g, 148.43 mmol, 20.57 mL, 2 eq), P(o-tolyl)₃(6.78 g, 22.26 mmol, 0.30 eq), Pd₂(dba)₃ (6.80 g, 7.42 mmol, 0.10 eq)and methyl prop-2-enoate (25.56 g, 296.86 mmol, 26.62 mL, 4 eq) in turnat 25° C. under N₂. The resulting mixture was stirred at 80° C. for 13hours. The reaction mixture was added FLO 500 mL at 25° C., extractedwith EtOAc (500 mL×3). The combined organic layers were washed withsaturated brine 200 mL (200 mL×1), dried over dry Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=50:1 to 10:1). Compound 6.7 (24 g, 63.42 mmol, 85.46% yield) wasobtained as a white solid.

To a solution of compound 6.7 (24 g, 63.42 mmol, 1 eq) in MeOH (1.50 L)was added Pd—C under Ar₂. The suspension was degassed under vacuum andpurged with H₂ several times. The mixture was stirred under H₂ (50 psi)at 25° C. for 24 hours. Pd/C was filtered off by filtration. Thefiltrate was concentrated to dry. Compound 6.6 (22 g, 57.83 mmol, 91.19%yield) was obtained as a white solid.

To a mixture of compound 6.5 (6 g, 15.77 mmol, 1 eq) in MeOH (60 mL) wasadded NaOH (1.89 g, 47.31 mmol, 3 eq) in one portion at 25° C. Themixture was stirred at 25° C. for 17 h. The pH was adjusted to around 4by progressively adding citric acid. Then the solid was precipitate out,filtered to give compound 6.4 (4 g, 10.92 mmol, 69.22% yield) as a whitesolid. LCMS (ESI): m/z: [M−H] calcd for C₁₃H₁₈N₂O₄: 265; found 265;RT=0.859 min. ¹H NMR (400 MHz, methanol-d₄) δ ppm 1.45-1.59 (m, 7H)2.56-2.70 (m, 2H) 2.83-2.98 (m, 2H) 6.91 (dd, 0.7=5.18, 1.43 Hz, 1H)7.71 (d, J=0.66 Hz, 1H) 8.06 (d, J=5.07 Hz, 1H).

A solution of compound 6.4 (1 g, 2.63 mmol, 1 eq) was added in i-PrNH2(6.90 g, 116.73 mmol, 10 mL, 44.38 eq) at 25° C. The mixture was addedAlMe₃ (568.79 mg, 7.89 mmol, 3 mL, 3 eq) dropwised at 25° C. Theresulting mixture was stirred at 25° C. for 23 hours. The reactionmixture was filtered and concentrated under reduced pressure to givecompound 6.3 (677 mg, 1.66 mmol, 63.17% yield).

To a solution of compound 6.3 (670 mg, 2.18 mmol, 1 eq) in THF (10 mL)was added a solution of BH₃-Me₂S (10 M, 872 μL, 4 eq) dropwise with at0° C. over a period of 1 mins under N₂. Then stirring at 25° C. 4 hours.The reaction was quenched by addition of 6 ml methanol, then thereaction mixture was concentrated under reduced pressure to removesolvent. The mixture was further purification by pre-HPLC to givecompound 6.2 (70 mg, 238.58 μmol, 10.94% yield) (70 mg, 238.58 μmol,10.94% yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd forC₁₆H₂₇N₃O₂: 294; found 294; RT=0.735 min.

To a mixture of compound 6.2 (70 mg, 177.88 μmol, 1 eq) in HCl/EtOAc (2mL) at 25° C. for 13 hours. The reaction mixture was concentrated underreduced pressure to give compound 6.1 (65 mg, crude) as light yellowoil.

To a mixture of compound 6.1 (63 mg, 236.66 μmol, 1 eq) and BrCN (25.07mg, 236.66 μmol, 17.41 μL, 1 eq) in THF (3 mL) was added DIEA (122.34mg, 946.64 μmol, 165.32 μL, 4 eq) in one portion at 0° C. under N₂. Themixture was stirred at 25° C. for 10 mins. The aqueous phase wasextracted with ethyl acetate (10 mL×3). The combined organic phase waswashed with brine (3 mL×3), dried with anhydrous Na₂SO₄, filtered andconcentrated in vacuum. The residue was purified by prep-HPLC (neutralcondition) to give 6 (10 mg, 45.81 μmol, 19.36% yield) as a yellow oil.LCMS (ESI): m/z: [M+H] calcd for C₁₂H₁₈N₄: 219; found 219; RT=2.279 min.¹H NMR (400 MHz, chloroform-d) δ ppm 1.25-1.28 (d, J=6.0 6 H) 1.90-2.10(m, 2H) 2.50-2.62 (m, 2H) 2.96-2.99 (m, 2H) 3.08-3.13 (m, 1H) 4.37 (s,2H) 6.35 (s, 1H) 6.4-6.50 (d, J=5.2 6 H) 7.98-7.99 (d, J=5.2 1H).

Example 7. Preparation of3-((2-aminopyridin-4-yl)oxy)azetidine-1-carbonitrile (7)

To a mixture of compound 7.4 (2 g, 11.55 mmol, 1 eq) in DMSO (40 mL) wasadded NaH (554.24 mg, 23.09 mmol, 2 eq) in one portion at 25° C. underN₂. The mixture was stirred at 25° C. for 2 hours, then added compound7.5 (2.97 g, 23.09 mmol, 2 eq) to mixture and heated to 90° C. andstirred for 15 hours. The mixture was cooled to 25° C. and poured intoice-water (50 mL). The aqueous phase was extracted with ethyl acetate(40 mL×3). The combined organic phase was washed with brine (30 mL×1),dried with anhydrous Na₂SO₄ concentrated in vacuum to give a crudeproduct as a brown solid. Then the crude product was purified by silicagel chromatography eluted with EtOAc:DCM=1:1 to give compound 7.2 (230mg, 90%) as a brown oil. ¹H NMR (400 MHz, chloroform-d) ppm 1.46 (s, 9H)4 (dd, J=9.79, 3.76 Hz, 2H) 4.33 (dd, J=9.54, 6.53 Hz, 2H) 4.91 (t,J=3.76 Hz, 1H) 5.91-6.01 (m, 1H) 6.18 (dd, J=6.02, 1.51 Hz, 1H) 7.84 (d,J=6.02 Hz, 1H). LCMS (ESI): m/z: [M+H] calcd for C₁₃H₁₉N₃CO₃: 266; found266; RT=1.547 min.

Compound 7.2 (230 mg, 866.91 μmol, 1 eq) was dissolved DCM (5 mL) andTFA (1 mL) at 25° C. under N₂. The mixture was stirred at 25° C. for 15hours. The mixture was added small amount of water and acetonitrile,then work up it by lyophilization to give compound 7.1 (300 mg, 724.75μmol, 83.60% yield, 95% purity, 2 TFA) as brown oil. The crude productwas used for next step directly without purification. ¹H NMR (400 MHz,methanol-d4) ppm 4.27 (dd, J=12.55, 4.52 Hz, 2H) 4.63 (dd, J=12.80, 6.78Hz, 2H) 5.25-5.42 (m, 1H) 6.27 (d, J=2.51 Hz, 1H) 6.58 (dd, J=7.28, 2.26Hz, 1H) 7.81 (d, J=7.53 Hz, 1H). LCMS (ESI): m/z: [M+H] calcd forC8H11N3.O2(C2HF302):166; found 166; RT=0.214 min.

The mixture of compound 7.1 (50 mg, 302.68 μmol, 1 eq), TEA (30.63 mg,302.68 μmol, 41.96 μL, 1 eq) and DIEA (117.36 mg, 908.04 μmol, 158.59μL, 3 eq) in DCM (5 mL) was added dropwise solution of BrCN (32.06 mg,302.68 μmol, 22.26 μL, 1 eq). The mixture was stirred at 25° C. for 10min. The reaction mixture was quenched by addition H₂O 5 mL at 25° C.,and then extracted with DCM (5 mL×3). The combined organic layers werewashed with saturated brines (3 mL×1), dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give acrude product. The crude product was purified by prep-HPLC (TFAcondition) to give the product 7 (13 mg, 68.35 μmol, 22.58% yield) as awhite solid. ¹H NMR (400 MHz, chloroform-d) ppm 4.27 (dd, J=8.82, 4.41Hz, 2H) 4.44 (br. s., 2H) 4.48-4.59 (m, 2H) 4.98 (br. s., 1H) 5.77 (s,1H) 6.02-6.15 (m, 1H) 7.94 (d, J=5.73 Hz, 1H). LCMS (ESI): m/z: [M+H]calcd for C₉H₁₀N₄O:191; found 191; RT=1.014 min.

Example 8. Preparation of4-((2-aminopyridin-4-yl)oxy)piperidine-1-carbonitrile (8)

To a solution of compound 8.4 (500 mg, 4.54 mmol, 1 eq) in DCM (15 mL)was added compound 8.3 (913.90 mg, 4.54 mmol, 1 eq) PPh₃ (1.79 g, 6.81mmol, 1.50 eq) and DIAD (1.38 g, 6.81 mmol, 1.32 mL, 1.50 eq) in turn at0° C. under N₂. The resulting mixture was stirred at 25° C. for 3 hours.The reaction mixture was added water 20 mL, extracted with EtOAc (20mL×3). The combined organic layers were washed with brine (20 mL×1),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1:1). Compound 8.2 (300 mg, 1.02 mmol,22.52% yield) was obtained as a white solid. ¹H NMR (400 MHz,chloroform-d) ppm 1.47 (s, 9H) 1.72-1.77 (m, 2H) 1.88-1.93 (m, 2H)3.33-3.37 (m, 2H) 3.64-3.68 (m, 2H) 4.37 (s, 2H) 4.47-4.51 (m, 1H) 5.98(d, J=2.0 Hz, 1H) 6.25 (dd, J=6.0 Hz, 2.0 Hz, 1H) 7.90 (d, J=6.0 Hz,1H).

A mixture of compound 8.2 (200 mg, 681.76 μmol, 1 eq) in EtOAc HCl (10mL) was stirred at 25° C. for 16 hours. The reaction mixture wasfiltered, the cake was washed with EtOAc (10 mL). The solid was removedsolvent under reduced pressure. Compound 8.1 (100 mg, 435.33 μmol,63.85% yield) was obtained as a white solid. ¹H NMR (400 MHz, D₂O-d4)ppm 2.02-2.13 (m, 4H) 3.18-3.36 (m, 4H) 4.84-4.85 (m, 1H) 6.33 (d, J=2.0Hz, 1H) 6.49 (dd, J=7.2 Hz, 2.0 Hz, 1H) 7.69 (m, 1H).

To a solution of compound 8.1 (50 mg, 217.67 μmol, 1 eq) in THF (5 mL)was added DIEA (28.13 mg, 217.67 μmol, 38.02 μL, 1 eq) and BrCN (23.06mg, 217.67 μmol, 16.01 μL, 1 eq) in turn at 0° C. under N₂. Theresulting mixture was stirred at 0° C. for 2.5 hours. The reactionmixture was added water 5 mL, extracted with EtOAc (10 mL×3). Thecombined organic layers were washed with brine (5 mL×1), dried overNa₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by prep-HPLC (basic condition).Product 8 (6.50 mg, 29.78 μmol, 13.68% yield) was obtained as a whitesolid. LCMS (ESI): m/z: [M+H] calcd for C₁₁H₁₄N₄O:218; found 219;RT=1.934 min. ¹H NMR (400 MHz, chloroform-d) ppm 1.95-2.03 (m, 4H)3.23-3.48 (m, 4H) 4.38 (s, 2H) 4.56 (m, 1H) 5.96 (s, 1H) 6.24 (s, 1H)7.93 (d, J=5.2 Hz, 1H).

Example 9. Preparation of3-((2-aminopyridin-4-yl)oxy)piperidine-1-carbonitrile (9)

To a mixture of compound 9.3 (2 g, 9.94 mmol, 1 eq) in DMSO (40 mL) wasadded NaH (476.99 mg, 19.88 mmol, 2 eq) in one portion. The mixture wasstirred under N₂ at 25° C. for 2 hours, then added compound 9.4 (2.56 g,19.88 mmol, 2 eq) to the mixture and heated to 90° C. and stirred for 15hours. The mixture was cooled to 25° C. and poured into ice-water (50mL). The aqueous phase was extracted with ethyl acetate (40 mL×3). Thecombined organic phase was washed with brine (30 mL×1), dried withanhydrous Na₂SO₄ concentrated in vacuum to give a crude product as abrown solid. Then the crude product was purified by silica gelchromatography eluted with (EtOAc:DCM=1:1) to give compound 9.2 (130 mg,420.98 μmol, 4.24% yield, 95% purity) as a brown oil. ¹H NMR (400 MHz,methanol-d₄) δ ppm 1.17-1.49 (m, 9H) 1.49-1.62 (m, 1H) 1.74-2.06 (m, 3H)2.94-3.15 (m, 1H) 3.32-4.14 (m, 3H)4.58 (br. s., 1H) 6.32 (br. s., 1H)6.46 (d, J=5.29 Hz, 1H) 7.59-7.81 (m, 1H). LCMS (ESI): m/z: [M+H] calcdfor C₁₅H₂₃N₃O₃: 294; found 294; RT=1.046 min.

Compound 9.2 (130 mg, 443.14 μmol, 1 eq) was dissolved HCl/EtOAc (10 mL)and MeOH (5 mL) at 25° C. under N₂. The mixture was stirred at 25° C.for 5 h. The mixture was concentrated in vacuum to give compound 9.1 (80mg, 393.27 μmol, 88.75% yield, 95% purity) as yellow oil. The crudeproduct was used for next step directly without purification. ¹H NMR(400 MHz, methanol-d4) δ ppm 1.79-1.91 (m, 1H) 1.94-2.15 (m, 3H) 3.17(br. s., 1H) 3.33 (br. s., 1H) 3.39-3.63 (m, 3H) 5.03 (br. s., 1H) 6.49(d, J=2.21 Hz, 1H) 6.62 (dd, J=7.28, 2.43 Hz, 1H) 7.77 (d, J=7.06 Hz,1H).

To a solution of compound 9.1 (80 mg, 413.97 μmol, 1 eq) in THF (3 mL)and TEA (125.67 mg, 1.24 mmol, 172.15 μL, 3 eq) was added a solution ofBrCN (43.85 mg, 413.97 μmol, 30.45 μL, 1 eq) in THF (3 mL) drop-wise at0° C. under N₂. The reaction mixture was stirred at 0° C. for 4 h. Tothe reaction mixture was added 4 mL water at 0° C. The aqueous phase wasextracted with ethyl acetate (15 mL×3). The combined organic phase waswashed with brine (5 mL×1), dried with anhydrous Na₂SO₄, filtered andconcentrated in vacuum. The crude product was purified by prep-TLC(neutral condition) to give product 9 (12 mg, 50.58 μmol, 12.22% yield,92% purity) as brown oil. ¹H NMR (400 MHz, methanol-d4) ppm 1.59-1.72(m, 1H) 1.88-1.97 (m, 3H) 3.34-3.41 (m, 1H) 3.42-3.51 (m, 1H) 4.62-4.72(m, 1H) 5.09-5.20 (m, 1H) 6.22-6.32 (m, 1H) 6.38-6.56 (m, 1H) 7.65-7.80(m, 1H). LCMS (ESI): m/z: [M+H] calcd for C₁₁H₁₄N₄O:219; found 219;RT=1.148 min.

Example 10. Preparation of3-(((2-aminopyridin-4-yl)oxy)methyl)azetidine-1-carbonitrile (10)

To a solution of compound 10.4 (1.50 g, 13.62 mmol, 1 eq) in DCM (15 mL)was added compound 10.3 (2.55 g, 13.62 mmol, 1 eq), PPh₃ (5.36 g, 20.43mmol, 1.50 eq) and DIAD (4.13 g, 20.43 mmol, 3.97 mL, 1.50 eq) in turnat 0° C. under N₂. The resulting mixture was stirred at 25° C. for 10hours. The reaction mixture was added water 5 mL, extracted with EtOAc(10 mL×3). The combined organic layers were washed with brine (5 mL×1),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1:1). Compound 10.2 (800 mg, 2.86 mmol,21.03% yield) was obtained as a white solid. ¹H NMR (400 MHz,chloroform-d) ppm 1.46 (s, 9H) 2.92-3 (m, 1H) 3.75-3.79 (m, 2H)4.06-4.09 (m, 4H) 4.39 (s, 2H) 5.98 (d, J=2.0 Hz, 1H) 6.26 (dd, J=6.0Hz, 2.0 Hz, 1H) 7.90 (d, J=5.6 Hz, 1H)

To a solution of compound 10.2 (300 mg, 1.07 mmol, 1 eq) in DCM (5 mL)was added TFA (1.54 g, 13.50 mmol, 1 mL, 12.62 eq) at 25° C. under N₂.The resulting mixture was stirred at 25° C. for 16 hours. The reactionmixture was concentrated under reduced pressure to give a residue andfreezing dry. Compound 10.1 (500 mg, crude) was obtained as a whitesolid. ¹H NMR (400 MHz, D₂O-d4) ppm 3.36-3.43 (m, 1H) 4.40-4.12 (m, 2H)4.22-4.31 (m, 4H) 6.42 (d, J=2.8 Hz, 1H) 6.62 (dd, J=7.6 Hz, 2.4 Hz, 1H)7.69 (d, J=6.8 Hz, 1H)

To a solution of compound 10.1 (300 mg, 736.61 μmol, 1 eq) in THF (5 mL)was added DIEA (285.60 mg, 2.21 mmol, 385.94 μL, 3 eq) and BrCN (78.02mg, 736.61 μmol, 54.18 μL, 1 eq) in turn at 0° C. under N₂. Theresulting mixture was stirred at 0° C. for 1 hour. The reaction mixturewas added water 10 mL, extracted with EtOAc (10 mL×3). The combinedorganic layers were washed with brine (20 mL×1), dried over Na₂SO₄,filtered and concentrated under reduced pressure to give a residue. Theresidue was purified by p-TLC (SiO₂, Ethyl acetate=0:1). Product 10 (14mg, 68.55 μmol, 9.31% yield) was obtained as a white solid. LCMS (ESI):m/z: [M+H] calcd for C₁₀H₁₂N₄O:204; found 205; RT=1.750 min. ¹H NMR (400MHz, chloroform-d) ppm 3.13-3.19 (m, 1H) 4.08-4.11 (m, 4H) 4.31-4.35 (m,2H) 4.96 (s, 2H) 6.05 (d, J=2.0 Hz, 1H) 6.30 (dd, J=6.0 Hz, 2.0 Hz, 1H)7.88 (d, J=6.0 Hz, 1H).

Example 11. Preparation of2-(((2-aminopyridin-4-yl)oxy)methyl)azetidine-1-carbonitrile (11)

To a mixture of compound 11.4 (500 mg, 2.67 mmol, 1 eq) and TEA (540.35mg, 5.34 mmol, 740.21 μL, 2 eq) in DCM (10 mL) was added MsCl (367.02mg, 3.20 mmol, 247.99 μL, 1.20 eq) dropwise at 0° C. under N₂. Thereaction mixture was diluted with H₂O 10 mL and extracted with DCM 10 mL(10 mL×3). The combined organic layers were washed with brine 20 mL (20mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give compound 11.3 (850 mg, crude) as a yellow oil.(Combined with a second batch to afford a total of 1.3 g). LCMS (ESI):m/z: [M+H] calcd for C₁₀H₁₉NSO₅: 265; found 166,210; RT=0.657, 0.693min.

To a mixture of compound 11.3 (1 g, 3.77 mmol, 1 eq) and compound 11.5(414.99 mg, 3.77 mmol, 1 eq) in acetonitrile (15 mL) was added K₂CO₃(1.04 g, 7.54 mmol, 2 eq) in one portion at 25° C. under N₂. Then heatedto 90° C. and stirred for 10 hours. The reaction mixture was dilutedwith H₂O 15 mL and extracted with EA 10 mL (10 mL×3). The combinedorganic layers were washed with brine 20 mL (20 mL×1), dried overNa₂SO₄, filtered and concentrated under reduced pressure to give aresidue. Then the residue was purified by column (SiO₂, DCM/MeOH=10:1)to give compound 11.2 (200 mg, 716 μmol, 18.99% yield) as a white solid.¹H NMR (400 MHz, chloroform-d) ppm 1.42 (s, 9H) 1.45 (s, 18H) 1.78 (br.s., 3H) 2.17-2.41 (m, 6H) 2.98-3.12 (m, 6H) 3.73-3.93 (m, 6H) 4.21-4.33(m, 3H) 4.35-4.63 (m, 6H) 6.03 (d, J=1.51 Hz, 1H) 6.30 (dd, J=6.02, 2.01Hz, 1H) 7.89 (d, J=6.02 Hz, 1H). LCMS (ESI): m/z: [M+H] calcd forC₁₄H₁₁N₃O₃: 279; found 187; RT=0.576, 0.630 min.

To a solution of compound 11.2 (200 mg, 716 μmol, 1 eq) in DCM (5 mL)was added TFA (1.54 g, 13.51 mmol, 1 mL, 18.86 eq) in one portion at 25°C. under N₂. The mixture was stirred at 25° C. for 10 hours. Thereaction mixture was concentrated under reduced pressure to givecompound 11.1 (240 mg, crude) as a light yellow solid. LCMS (ESI): m/z:[M+H] calcd for C₉H₁₃N₃O: 179; found 180; RT=0.175, 0.240 min.

To a mixture of compound 11.1 (50 mg, 278.99 μmol, 1 eq) and DIPEA(144.23 mg, 1.12 mmol, 194.91 μL, 4 eq) in DCM (2 mL) was added asolution of carbononitridic bromide (29.55 mg, 278.99 μmol, 20.52 μL, 1eq) in 0.5 ml DCM dropwise at 0° C. under N₂. The mixture was stirred at0° C. for 30 mins. The reaction mixture was diluted with H₂O 2 mL andextracted with DCM 2 mL (2 mL×3). The combined organic layers werewashed with brine 3 mL (3 mL×1), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. Then, the residuewas purified by prep-HPLC (TFA condition) to give product 11 (10 mg,48.96 μmol, 17.55% yield) as a yellow solid. ¹H NMR (400 MHz,chloroform-d) ppm 2.32-2.43 (m, 1H) 2.43-2.54 (m, 1H) 4.06-4.25 (m, 3H)4.43 (br. s., 1H) 4.64-4.77 (m, 1H) 6.03 (d, J=2.01 Hz, 1H) 6.30 (dd,J=5.52, 2.01 Hz, 1H) 7.94 (d, J=6.02 Hz, 1H). LCMS (ESI): m/z: [M+H]calcd for C₁₀H₁₂N₄O: 204; found 205; RT=1.754 min.

Example 12. Preparation of2-(((2-aminopyridin-4-yl)oxy)methyl)piperidine-1-carbonitrile (12)

Compound 12.7 (2 g, 9.29 mmol, 1 eq) was added into HCl/EtOAc (20 mL),the reaction was stirred for 15 hours at 18° C. The reaction mixture wasconcentrated under reduced pressure to give compound 12.6 (2.10 g,crude) as a white solid.

To a mixture of compound 12.6 (2.10 g, 11.16 mmol, 1 eq, 2HCl) and TEA(3.39 g, 33.48 mmol, 4.64 mL, 3 eq) in DCM (25 mL) was added compound12.5 (5.19 g, 23.44 mmol, 2.10 eq) in one portion at 0° C. The mixturewas then heated to 18° C. and stirred for 15 hours. The reaction mixturewas quenched by addition H₂O 20 mL and then diluted with DCM 10 mL andextracted with DCM 20 mL (20 mL×3). The combined organic layers werewashed with brine 40 mL (40 mL×1), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give compound 12.4 (4.50 g,crude) as a yellow solid. LCMS (ESI): m/z: [M−H] calcd for C₁₈H₁₉N₃O₉S₂:485; found 486; RT=0.869 min.

To a mixture of compound 12.4 (4.50 g, 9.27 mmol, 1 eq) and compound12.3 (1.22 g, 11.12 mmol, 1.20 eq) in DMF (50 mL) was added K₂CO₃ (2.56g, 18.54 mmol, 2 eq) in one portion at 18° C. under N₂. Then heated to80° C. and stirred for 15 hours. The reaction mixture was diluted withH₂O 50 mL and extracted with EtOAc 120 mL (40 mL×3). The combinedorganic layers were washed with brine 80 mL (80 mL×1), dried overNa₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by prep-TLC (SiO₂, DCM:MeOH=10:1) togive compound 12.2 (260 mg, 662.54 μmol, 7.15% yield) as a yellow solid.LCMS (ESI): m/z: [M−H] calcd for C₁₇H₂₀N₄O₅S: 392; found 393; RT=0.714min.

To a mixture of compound 12.2 (260 mg, 662.54 μmol, 1 eq) and K₂CO₃(366.28 mg, 2.65 mmol, 4 eq) in DMF (2 mL) was added THIOPHENOL (146 mg,1.33 mmol, 135.18 μL, 2 eq) dropwise at 18° C. under N₂. The mixture wasstirred at 18° C. for 4 hours. The residue was purified by prep-HPLC(TFA condition) to give compound 12.1 (80 mg, 385.97 μmol, 58.26% yield)as a white solid. LCMS (ESI): m/z: [M−H] calcd for C₁₁H₁₇N₃O: 207; found208; RT=1.039 min. ¹H NMR (400 MHz, methanol-d₄) δ ppm 1.60-1.83 (m, 2H)1.91-2.10 (m, 2H) 3.04-3.18 (m, 1H) 3.49 (br d, J=12.80 Hz, 1H) 3.65(td, J=7.37, 3.45 Hz, 1H) 4.27 (dd, J=10.79, 7.28 Hz, 1H) 4.41 (dd,J=10.79, 3.39 Hz, 1H) 6.45 (d, J=2.51 Hz, 1H) 6.62 (dd, J=7.28, 2.51 Hz,1H) 7.79 (d, J=7.28 Hz, 1H)

To a mixture of compound 12.1 (70 mg, 337.72 μmol, 1 eq) and DIEA(174.59 mg, 1.35 mmol, 235.93 μL, 4 eq) in DMF (3 mL) was addedcarbononitridic bromide (35.77 mg, 337.72 μmol, 24.84 μL, 1 eq) in oneportion at 0° C. under N₂. The mixture was stirred at 0° C. for 30 mins.The reaction mixture diluted with H₂O 3 mL and extracted with DCM 9 mL(3 mL×3). The combined organic layers were washed with brine 4 mL (4mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by prep-HPLC(neutral condition) to give product 12 (15 mg, 64.58 μmol, 19.12% yield)as a yellow solid. LCMS (ESI): m/z: [M−H] calcd for C₁₂H₁₆N₄O: 232;found 233; RT=1.970 min. ¹H NMR (400 MHz, DMSO-d) δ ppm 1.48 (br d,J=8.03 Hz, 2H) 1.60 (br s, 1H) 1.74 (br d, J=6.65 Hz, 2H) 3.10-3.19 (m,1H) 3.43 (br s, 1H) 3.54 (br s, 1H) 4.29 (d, J=4.89 Hz, 2H) 6.38 (d,J=2.38 Hz, 1H) 6.57 (dd, J=7.28, 2.51 Hz, 1H) 7.83 (br s, 2H) 7.87 (d,J=7.28 Hz, 1H)

Example 13. Preparation ofN-(2-((2-aminopyridin-4-yl)oxy)ethyl)-N-methylcyanamide (13)

To a mixture of compound 13.4 (2 g, 11.41 mmol, 1 eq) and CBr₄ (5.68 g,17.11 mmol, 1.50 eq) in THF (20 mL) was added PPh₃ (4.49 g, 17.11 mmol,1.50 eq) in one portion at 18° C. under N₂. The mixture was stirred at18° C. for 1 hour. The reaction mixture was filtered and concentratedunder reduced pressure to give a residue. The residue was purified bycolumn chromatography (SiO₂, DCM) to give compound 13.3 (1.80 g, 7.56mmol, 66.25% yield) as a colorless oil. ¹H NMR (400 MHz, chloroform-d)ppm 1.31-1.47 (m, 9H) 2.80-2.91 (m, 3H) 3.37 (br. s., 2H) 3.46-3.59 (m,2H).

To a solution of compound 13.5 (832.35 mg, 7.56 mmol, 1 eq) in DMF (2mL) was added NaH (362.84 mg, 15.12 mmol, 2 eq) portionwise at 20° C.under N₂. The mixture was stirred at 20° C. for 0.5 hour. Then themixture was added compound 13.3 (1.80 g, 7.56 mmol, 1 eq) in one portionand heated to 90° C. stirred for 14.5 hours. The reaction mixture wasdiluted with H₂O 3 mL and extracted with EA 2 mL (2 mL×3). The combinedorganic layers were washed with brine 5 mL (5 mL×1), dried over Na₂SO₄,filtered and concentrated under reduced pressure to give compound 13.2(1 g, crude) as a yellow oil. ¹H NMR (400 MHz, methanol-d4) δ ppm1.37-1.56 (m, 9H) 2.86 (s, 4H) 2.94 (br. s., 2H) 2.99 (s, 5H) 3.54-3.68(m, 1H) 4.01-4.16 (m, 2H) 6.10 (d, J=2.21 Hz, 1H) 6.18-6.32 (m, 1H) 7.70(d, J=5.73 Hz, 1H) 7.98 (s, 1H). LCMS (ESI): m/z: [M+H] calcd forC₁₃H₂₁N₃O3: 267; found 268; RT=0.631, 0.833 min.

Compound 13.2 (500 mg, 1.87 mmol, 1 eq) was added into a solution ofHCl/MeOH (8 mL). The mixture was stirred at 18° C. for 15 hours. Thereaction mixture was concentrated under reduced pressure to givecompound 13.1 (300 mg, 1.79 mmol, 95.94% yield) as a yellow solid. LCMS(ESI): m/z: [M+H] calcd for C₈H₁₃N₃O: 167; found 168; RT=0.166, 0.258min.

To a mixture of compound 13.1 (150 mg, 897.08 μmol, 1 eq) and DIEA(463.75 mg, 3.59 mmol, 626.69 μL, 4 eq) in DCM (3 mL) was addedcarbononitridic bromide (95.02 mg, 897.08 μmol, 65.98 μL, 1 eq) in oneportion at 0° C. under N₂. The mixture was stirred at 0° C. for 30 mins.The reaction mixture diluted with H₂O 3 mL and extracted with DCM 3 mL(3 mL×3). The combined organic layers were washed with brine 4 mL (4mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by prep-HPLC(neutral condition) to give product 13 (10 mg, 52.02 μmol, 5.80% yield)as a yellow solid. ¹H NMR (400 MHz, DMSO-d6) δ ppm 2.83-2.93 (m, 3H)3.37 (t, J=4.85 Hz, 2H) 4.06-4.15 (m, 2H) 5.90 (br. s., 2H) 5.99 (d,J=2.21 Hz, 1H)6.11-6.21 (m, 1H) 7.74 (d, J=6.17 Hz, 1H). LCMS (ESI):m/z: [M+H] calcd for C₉H₁₄N₄O: 192; found 193; RT=0.508 min.

Example 14. Preparation ofN-(1-((2-aminopyridin-4-yl)oxy)propan-2-yl)-N-methylcyanamide (14)

To a solution of compound 14.7 (1 g, 9.08 mmol, 1 eq) in THF (20 mL) wasadded Boc₂O (5.95 g, 27.24 mmol, 6.26 mL, 3 eq), TEA (3.68 g, 36.32mmol, 5.03 mL, 4 eq) and DMAP (332.79 mg, 2.72 mmol, 0.30 eq) at 25° C.under N₂. The resulting mixture was stirred at 25° C. for 16 hours. Thereaction mixture was added water (30 mL), extracted with EtOAc (50mL×3). The organic phase was separated, washed with saturated NaCl (30mL) and dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=20:1 to 3:1).Compound 14.6 (1.50 g, 3.65 mmol, 40.25% yield) was obtained as a yellowsolid.

To a solution of compound 14.6 (1.50 g, 3.65 mmol, 1 eq) in MeOH (20 mL)was added NaOH (438 mg, 10.95 mmol, 3 eq) at 25° C. under N₂. Theresulting mixture was stirred at 25° C. for 16 hours. The reactionmixture was added water (30 mL), extracted with EtOAc (50 mL 15×3). Theorganic phase was separated, washed with saturated NaCl (30 mL) anddried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=20:1 to 3:1). Compound 14.5 (550 mg, 2.62mmol, 71.68% yield) was obtained as a white solid.

To a mixture of compound 14.5 (500 mg, 2.38 mmol, 1 eq) and compound14.4 (330.30 mg, 3.57 mmol, 1.50 eq) in DMSO (20 mL) was added K₂CO₃(657.88 mg, 4.76 mmol, 2 eq) in one portion at 20° C. under N₂. Themixture was stirred at 20° C. for 15 hours. The aqueous phase wasextracted with EtOAc (20 mL×3). The combined organic phase was washedwith brine (5 mL), dried with anhydrous Na₂SO₄, filtered andconcentrated in vacuum. Compound 14.3 (900 mg, crude) was obtained as acolorless oil. ¹H NMR (400 MHz, chloroform-d) ppm 1.54 (s, 9H), 2.29 (s,3H), 4.63 (s, 2H), 6.52 (dd, 7=5.6 Hz, 2.0 Hz, 1H), 7.58 (d, J=2.0 Hz,1H), 8.13 (d, J=6.0 Hz, 1H), 9.12 (s, 1H).

To a mixture of compound 14.3 (360 mg, 1.35 mmol, 1 eq) and methanamine(2 M, 1.35 mL, 2 eq) in EtOH (2 mL) was added CH₃COOH (40.59 mg, 675.95μmol, 38.66 μL, 0.50 eq) and NaBH₃CN (212.38 mg, 3.38 mmol, 2.50 eq) inone portion at 20° C. under N₂. The mixture was stirred at 20° C. for 20hours. The mixture was diluted with water (5 mL), neutralized with solidNaHCO₃ until no CO2 was evolved. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=10:1 to 0:1).Compound 14.2 (110 mg, 390.97 μmol, 28.96% yield) was obtained as acolorless oil. ¹H NMR (400 MHz, chloroform-d) ppm 1.18 (d, J=6.4 Hz,3H), 1.53 (s, 9H), 2.49 (s, 3H), 3.03-3.06 (m, 1H), 3.89-4.02 (m, 2H),6.53 (dd, J=5.6 Hz, 2.4 Hz, 1H), 7.54 (d, J=2.4 Hz, 1H), 8.04 (d, J=6.0Hz, 1H).

To a solution of compound 14.2 (110 mg, 390.97 μmol, 1 eq) in HCl/EtOAc(20 mL) at 20° C. under N₂. The mixture was stirred at 20° C. for 15hours. Filtered and concentrated in vacuum. Filtered and concentrated invacuum. Compound 14.1 (110 mg, 378.50 μmol, 96.81% yield, 3HCl) wasobtained as a black brown solid. ¹H NMR (400 MHz, methanol-d4) ppm 1.46(d, J=6.8 Hz, 3H), 2.78 (s, 3H), 2.73-2.75 (m, 1H), 2.49 (s, 3H),4.29-4.49 (m, 2H), 6.46 (d, 0.7=2.4 Hz, 1H), 6.63 (dd, J=2.4 Hz, J=1.2Hz, 1H), 7.78 (d, J=7.2 Hz, 1H).

To a mixture of compound 14.1 (80 mg, 441.43 μmol, 1 eq) andcarbononitridic bromide (46.76 mg, 441.43 μmol, 32.47 μL, 1 eq) in DMF(1 mL) was added DIEA (228.20 mg, 1.77 mmol, 308.38 μL, 4 eq) in oneportion at 0° C. under N₂. The mixture was stirred at 0° C. for 10 mins.The residue was purified by prep-HPLC (basic condition). Product 14 (10mg, 48.49 μmol, 10.98% yield) was obtained as a black brown solid. ¹HNMR (400 MHz, DMSO-d6) ppm 1.22 (d, J=6.8 Hz, 3H), 2.87 (s, 3H),3.43-3.45 (m, 1H), 3.89-4.02 (m, 2H), 5.83 (s, 2H), 5.97 (d, J=2.4 Hz,1H), 6.16 (dd, J=2.0 Hz, J=5.6 Hz, 1H), 7.73 (d, J=6.0 Hz, 1H). LCMS(ESI): m/z: [M+H] calcd for C₁₀H₁₄N₄O: 207; found 207; RT=1.842 min.

Example 15. Preparation ofN-(2-((2-aminopyridin-4-yl)oxy)propyl)-N-methylcyanamide (15)

The solution of compound 15.6 (1 g, 17.22 mmol, 1.20 mL, 1 eq) in MeNH₂(ethanol solution) (534.80 mg, 17.22 mmol, 3 mL, 1 eq) was stirred at70° C. for 15 hour. The mixture was concentrated under reduced pressureto give a residue. The crude product compound 15.5 (1 g, 11.22 mmol,65.15% yield) was used into the next step without further purification.¹H NMR (400 MHz, chloroform-d) ppm 1.05-1.16 (m, 3H) 2.27-2.46 (m, 4H)2.57 (dd, J=12.13, 2.87 Hz, 1H) 3.79 (d, J=6.17 Hz, 1H).

To a solution of compound 15.5 (1 g, 11.22 mmol, 1 eq) in THF (10 mL)was added TEA (2.27 g, 22.44 mmol, 3.11 mL, 2 eq) and Boc₂O (3.67 g,16.83 mmol, 3.87 mL, 1.50 eq). The mixture was stirred at 25° C. for 15hours. The reaction mixture was quenched by addition H₂O 15 mL at 25°C., and extracted with DCM (20 mL×3). The combined organic layers werewashed with saturated brines (15 mL×1), dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give acrude product. The crude product was purified by column chromatography(SiO₂, Petroleum: ether/Ethyl acetate=2:1) to give compound 15.4 (1.30g, 6.87 mmol, 61.22% yield) as a yellow oil. ¹H NMR (400 MHz,chloroform-d) ppm 1.15 (d, J=6.17 Hz, 3H) 1.36-1.54 (m, 9H) 2.91 (s, 3H)3.03-3.43 (m, 2H) 3.88-4.13 (m, 1H).

To a solution of compound 15.4 (1.20 g, 6.34 mmol, 1 eq) and compound15.3 (698.19 mg, 6.34 mmol, 1 eq) in DCM (20 mL) was added PPh₃ (2.49 g,9.51 mmol, 1.50 eq) and DIAD (1.92 g, 9.51 mmol, 1.85 mL, 1.50 eq). Themixture was stirred at 25° C. for 15 hours. The reaction mixture wasquenched by addition H₂O 25 mL at 25° C., and extracted with DCM (20mL×3). The combined organic layers were washed with saturated brines (15mL×1), dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure to give a crude product. The crude product waspurified by column chromatography (SiO₂, Petroleum:ether/Ethylacetate=1:1) to give compound 15.2 (200 mg, crude) as a white solid. ¹HNMR (400 MHz, chloroform-d) δ ppm 1.21-1.31 (m, 4H) 1.34 (d, J=6.02 Hz,6H) 1.46 (br. s., 10H) 2.05 (s, 1H) 2.75-3.05 (m, 3H) 4.13 (d, J=7.53Hz, 1H) 4.35 (br. s., 3H) 4.50-4.77 (m, 2H) 5.90-6.11 (m, 2H) 6.24 (dd,J=6.02, 2.01 Hz, 2H) 7.89 (d, J=6.02 Hz, 2H). LCMS (ESI): m/z: [M+H]calcd for C₁₄H₂₃N₃O₃: 282; found 282; RT=0.705 min.

A mixture of compound 15.2 (200 mg, 710.86 μmol, 1 eq) in HCl/EtOAc (5mL) was stirred at 25° C. for 1 hour. The reaction mixture concentratedunder reduced pressure to give a crude product compound 15.1 (120 mg,crude, 2 HCl) as a white solid. ¹H NMR (400 MHz, methanol-d₄) ppm 1.24(s, 2H) 1.39 (d, J=6.02 Hz, 4H) 1.45 (d, J=6.02 Hz, 3H) 2.78 (s, 3H)3.35-3.45 (m, 2H) 4.72-4.83 (m, 1H) 4.99-5.11 (m, 1H) 6.36 (s, 1H) 6.49(s, 1H) 6.52 (d, J=2.51 Hz, 1H) 6.63 (s, 1H) 7.65-7.73 (m, 1H) 7.78 (d,J=7.53 Hz, 1H). LCMS (ESI): m/z: [M+H] calcd for C₉H₁₅N₃O: 182; found182; RT=0.092 min.

To a solution of compound 15.1 (120 mg, 472.14 μmol, 1 eq, 2HCl) in DMF(1.50 mL) was added DIEA (244.08 mg, 1.89 mmol, 329.84 μL, 4 eq) andBrCN (50.01 mg, 472.14 μmol, 34.73 μL, 1 eq). The mixture was stirred at0° C. for 10 mins. The residue was purified by prep-HPLC (neutralcondition) to give the product 15 (3 mg, 14.55 μmol, 3.08% yield) as awhite solid. ¹H NMR (400 MHz, methanol-d₄) δ ppm 1.35 (d, J=6.02 Hz, 3H)2.96 (s, 3H) 3.23-3.32 (m, 2H) 4.72-4.82 (m, 1H) 6.16 (d, J=2.01 Hz, 1H)6.30 (dd, J=6.02, 2.51 Hz, 1H) 7.69-7.81 (m, 1H). LCMS (ESI): m/z: [M+H]calcd for C₁₄H₂₃N₃O₃: 207; found 207; RT=1.083 min.

Example 16. Preparation of2-(2-((2-aminopyridin-4-yl)oxy)ethyl)pyrrolidine-1-carbonitrile (16)

To a mixture of compound 16.6 (2 g, 8.72 mmol, 1 eq) in THF (20 mL) wasadded LAH (661.85 mg, 17.44 mmol, 2 eq) in one portion at 0° C. underN₂. The mixture was stirred at 0° C. for 2 hours. After the reactionmixture was cooled to 0° C., the reaction mixture was quenched byaddition of 5 mL of H₂O, followed by 2 mL of 15% aqueous NaOH. Afterbeing stirred at room temperature for 10 mins, the solid was removed byfiltration. The filtrate was concentrated to dryness to give crudeproduct. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=10/1 to 2:1). Compound 16.5 (1.38 g, 6.41mmol, 73.51% yield) was obtained as a colorless oil.

To a mixture of compound 16.5 (800 mg, 3.72 mmol, 1 eq) and carbontetrabromide (1.85 g, 5.58 mmol, 1.50 eq) in THF (15 mL) was added PPh₃(1.46 g, 5.58 mmol, 1.50 eq) in one portion at 0° C. under N₂. Themixture was stirred at 0° C. for 2 hours. The aqueous phase wasextracted with ethyl acetate (10 mL×3). The combined organic phase waswashed with brine (5 mL), dried with anhydrous Na₂SO₄, filtered andconcentrated in vacuum. The residue was purified by columnchromatography (SiO₂, Petroleum ether/ethyl acetate=20/1 to 5:1).Compound 16.4 (640 mg, 2.30 mmol, 61.84% yield) was obtained as acolorless oil. LCMS (ESI): m/z: [M+H−56] called for C₁₂H₂₀BrNO₂: 222;found 222; RT=0.103 min.

To a mixture of compound 16.4 (300 mg, 1.08 mmol, 1 eq) and compound16.3 (118.92 mg, 1.08 mmol, 1 eq) in DMF (10 mL) was added K₂CO₃ (298.53mg, 2.16 mmol, 2 eq) in one portion at 15° C. under N₂. The mixture wasstirred at 80° C. for 2 hours. The aqueous phase was extracted withethyl acetate (10 mL×3). The combined organic phase was washed withbrine (5 mL), dried with anhydrous Na₂SO₄, filtered and concentrated invacuum. Compound 16.2 (70 mg, crude) was obtained as black brown oil. Itwas combined with a second batch for a total of 140 mg, crude. LCMS(ESI): m/z: [M+H] called for C₁₆H₂₅N₃O₃: 308; found 308; RT=0.689 min.

A mixture of compound 16.2 (140 mg, 455.45 μmol, 1 eq) in HCl/EtOAc (10mL) was stirred at 15° C. for 15 hours. The mixture was concentrated invacuum to give crude product. Compound 16.1 (75 mg, 361.85 μmol, 79.45%yield) was obtained as a colorless oil. LCMS (ESI): m/z: [M+H] calledfor C₁₁H₁₇N₃O: 208; found 208; RT=0.102 min.

To a mixture of compound 16.1 (75 mg, 361.85 μmol, 1 eq) and DIEA (93.53mg, 723.69 μmol, 126.39 μL, 2 eq) in DMF (2 mL) was addedcarbononitridic bromide (38.33 mg, 361.85 μmol, 26.62 μL, 1 eq) in oneportion at 0° C. under N₂. The mixture was stirred at 15° C. for 15hours. The residue was purified by prep-HPLC (basic conditions). Product16 (2 mg, 8.61 μmol, 2.38% yield) was obtained as a white solid. ¹H NMR(400 MHz, methanol-d₄) δ ppm 1.57-1.79 (m, 1H) 1.84-2.29 (m, 5H) 2.92(br d, J=13.23 Hz, 1H) 3.38-3.58 (m, 2H) 3.83 (quin, J=6.73 Hz, 1H)4.02-4.26 (m, 2H) 6.10 (d, 0.7=2.21 Hz, 1H) 6.25 (dd, J=6.06, 2.32 Hz,1H) 7.70 (d, 0.7=6.17 Hz, 1H). LCMS (ESI): m/z: [M+H] called forC₁₂H₁₆N₄O: 233; found 233; RT=0.983 min.

Example 17. Preparation of3-(2-((2-aminopyridin-4-yl)oxy)ethyl)pyrrolidine-1-carbonitrile (17)

To a mixture of ethyl 2-diethoxyphosphorylacetate (20 g, 89.21 mmol,17.70 mL, 1 eq) and K₂CO₃ (30.82 g, 223.03 mmol, 2.50 eq) in DMF (100mL) was added compound 17.7 (19.83 g, 107.05 mmol, 1.20 eq) in oneportion at 20° C. under N₂. The mixture was stirred at 50° C. for 15hours. The aqueous phase was extracted with EtOAc (50 mL×3). Thecombined organic phase was washed with brine (5 mL), dried withanhydrous Na₂SO₄, filtered and concentrated in vacuum. The residue waspurified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=10:1). Compound 17.6 (7 g, 27.42 mmol, 30.73% yield) wasobtained as a colorless oil.

To a mixture of compound 17.6 (3 g, 11.75 mmol, 1 eq) in THF (60 mL) wasadded LAH (2.23 g, 58.75 mmol, 5 eq) in one portion at 0° C. under N₂.The mixture was stirred at 0° C. for 2 hours. After the reaction mixturewas cooled to 0° C., the reaction mixture was quenched by addition of 50mL of H₂O, followed by 10 mL of 15% aqueous NaOH. After being stirred atroom temperature for 10 mins, the solid was removed by filtration. Thefiltrate was concentrated to dryness to give crude product. The residuewas purified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=10:1 to 4:1). Compound 17.5 (1.07 g, 5.02 mmol, 42.70% yield)was obtained as a colorless oil.

To a solution of compound 17.5 (670 mg, 3.14 mmol, 1 eq) in MeOH (10 mL)was added Pd/C (200 mg, 10% purity) under N₂. The suspension wasdegassed under vacuum and purged with H₂ several times. The mixture wasstirred under H₂ (15 psi) at 15° C. for 3 hours. The reaction mixturewas filtered and the filter was concentrated. Compound 17.4 (500 mg,2.32 mmol, 73.96% yield) was obtained as a colorless oil.

To a mixture of compound 17.4 (400 mg, 1.86 mmol, 1 eq) andmethanesulfonyl chloride (319.24 mg, 2.79 mmol, 215.71 μL, 1.50 eq) inDCM (2 mL) was added TEA (376.01 mg, 3.72 mmol, 515.09 μL, 2 eq) in oneportion at 0° C. under N₂. The mixture was stirred at 0° C. for 2 hours.The aqueous phase was extracted with EtOAc (20 mL×3). The combinedorganic phase was washed with brine (5 mL), dried with anhydrous Na₂SO₄,filtered and concentrated in vacuum. Compound 17.3 (400 mg, crude) wasobtained as a colorless oil.

To a solution of compound 17.3a (149.75 mg, 1.36 mmol, 1 eq) in ACN (7mL) was added K₂CO₃ (375.93 mg, 2.72 mmol, 2 eq) and compound 17.3 (400mg, 1.36 mmol, 1 eq) at 25° C. under N₂. The resulting mixture wasstirred at 80° C. for 16 hours. The reaction mixture was added water (5mL), extracted with EtOAc (10 mL×3). The combined organic layers werewashed with brine (10 mL×1), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, DCM:MeOH=100:1 to 5:1).Compound 17.2 (200 mg, 650.64 μmol, 47.84% yield) was obtained as ayellow oil. ¹H NMR (400 MHz, chloroform-d) ppm 1.47-1.58 (m, 11H)1.84-1.89 (m, 2H) 2.04-2.06 (m, 1H) 2.34 (m, 1H) 2.93-2.98 (m, 1H)3.27-3.66 (m, 3H) 3.97-4 (m, 2H) 4.40 (s, 2H) 5.97 (s, 1H) 6.25 (d,J=5.6 Hz, 1H) 7.90 (d, J=5.6 Hz, 1H)

A solution of compound 17.2 (200 mg, 650.64 μmol, 1 eq) in HCl/EtOAc (5mL) was stirred at 25° C. for 1 h. The reaction mixture was concentratedunder reduced pressure to give a residue. Compound 17.1 (90 mg, 321.21μmol, 49.37% yield) was obtained as a yellow solid. ¹H NMR (400 MHz,methanol-d4) ppm 1.73-1.77 (m, 1H) 2.02-2.03 (m, 2H) 2.26-2.65 (m, 1H)2.52-2.54 (m, 1H) 2.94-2.97 (m, 1H) 3.42-3.61 (m, 4H) 4.22-4.26 (m, 2H)6.41 (d, J=2.4 Hz, 1H) 6.54 (dd, J=7.2 Hz, 2.0 Hz, 1H) 7.90 (d, J=7.6Hz, 1H).

To a solution of compound 17.1 (90 mg, 321.21 μmol, 1 eq) in DMF (3 mL)was added DIEA (166.05 mg, 1.28 mmol, 224.39 μL, 4 eq) and BrCN (34.02mg, 321.21 μmol, 23.63 μL, 1 eq) in turn at 0° C. under N₂. Theresulting mixture was stirred at 0° C. for 1 hour. The reaction mixturewas added water 10 mL, extracted with EtOAc (10 mL×3). The combinedorganic layers were washed with brine (20 mL×1), dried over Na₂SO₄,filtered and concentrated under reduced pressure to give a residue. Theresidue was purified by prep-HPLC (basic condition). Product 17 (10 mg,43.05 μmol, 13.40% yield) was obtained as a white solid. LCMS (ESI):m/z: [M+H] calcd for C₁₂H₁₆N₄O:232; found 233; RT=1.506 min. ¹H NMR (400MHz, chloroform-d) ppm 1.47-1.67 (m, 1H) 1.86-1.90 (m, 2H) 2.11-2.13 (m,1H) 2.35-2.46 (m, 1H)3.10-3.12 (m, 1H) 3.40-3.61 (m, 3H) 3.97-4.01 (m,2H) 4.40 (s, 2H) 5.95 (d, J=2.0 Hz, 1H) 6.23 (dd, J=6.0 Hz, 2.0 Hz, 1H)7.91 (d, J=5.6 Hz, 1H).

Example 18. Preparation of2-(3-((2-aminopyridin-4-yl)oxy)propyl)pyrrolidine-1-carbonitrile (18)

To a mixture of compound 18.12 (20 g, 92.92 mmol, 1 eq), EDCI (17.81 g,92.92 mmol, 1 eq) and HOBt (12.55 g, 92.92 mmol, 1 eq) in DMF (200 mL)was added DIEA (48 g, 92.92 mmol, 1 eq) and compound 18.11 (9.06 g,92.92 mmol, 1 eq) in one portion at 0° C. under N₂. The mixture wasstirred at 0° C. for 2 hours. The aqueous phase was extracted with EtOAc(50 mL×3). The combined organic phase was washed with brine (5 mL),dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. Theresidue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=3:1). Compound 18.10 (19.60 g, 75.88 mmol, 81.66%yield) was obtained as a black brown oil.

To a solution of compound 18.10 (10 g, 38.71 mmol, 1 eq) in THF (200 mL)was added DIBAL-H (6.01 g, 42.58 mmol, 1.10 eq) in one portion at −78°C. under N₂. The mixture was stirred at −78° C. for 2 hours. After thereaction mixture was cooled to 0° C., the reaction mixture was quenchedby addition of 100 mL of H₂O, followed by 15 mL of 15% aqueous NaOH.After being stirred at room temperature for 10 min, the solid wasremoved by filtration. The filtrate was concentrated to dryness to givecrude product. Compound 18.9 (6.70 g, crude) was obtained as colorlessoil.

To a mixture of compound 18.8 (4.69 g, 20.91 mmol, 4.15 mL, 1 eq) andK₂CO₃ (7.23 g, 52.27 mmol, 2.50 eq) in DMF (100 mL) was added compound18.9 (5 g, 25.09 mmol, 1.20 eq) in one portion at 20° C. under N₂. Themixture was stirred at 50° C. for 15 hours. The aqueous phase wasextracted with ethyl acetate (50 mL×3). The combined organic phase waswashed with brine (5 mL), dried with anhydrous Na₂SO₄, filtered andconcentrated in vacuum. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=10:1). Compound 18.7(3.20 g, 11.88 mmol, 56.82% yield) was obtained as a colorless oil.

To a solution of compound 18.7 (2 g, 7.43 mmol, 1 eq) in THF (30 mL) wasadded LAH (1.41 g, 37.15 mmol, 5 eq) in one portion at 0° C. under N₂.The mixture was stirred at 0° C. for 15 min. After the reaction mixturewas cooled to 0° C., the reaction mixture was quenched by addition of 5mL of H₂O, followed by 2 mL of 15% aqueous NaOH. After being stirred atroom temperature for 10 min, the solid was removed by filtration. Thefiltrate was concentrated to dryness to give crude product. The residuewas purified by prep-TLC (SiO₂, PE:ethyl acetate=2:1). Compound 18.6(1.08 g, 4.71 mmol, 63.39% yield) was obtained as a colorless oil. LCMS(ESI): m/z: [M+H] called for C₁₂H₂₃NO₃: 230; found 230; RT=0.741 min.

To a mixture of compound 18.6 (300 mg, 1.31 mmol, 1 eq) and TEA (198.84mg, 1.96 mmol, 272.38 μL, 1.50 eq) in DCM (10 mL) was added compound18.5 (180.07 mg, 1.57 mmol, 121.67 μL, 1.20 eq) in one portion at 0° C.under N₂. The mixture was stirred at 0° C. for 15 min. The aqueous phasewas extracted with ethyl acetate (30 mL×3). The combined organic phasewas washed with brine (5 mL), dried with anhydrous Na₂SO₄, filtered andconcentrated in vacuum. Compound 18.4 (600 mg, crude) was obtained aslight yellow oil.

To a mixture of compound 18.4 (600 mg, 1.95 mmol, 1 eq) and compound18.3 (214.91 mg, 1.95 mmol, 1 eq) in DMF (5 mL) was added K₂CO₃ (539.51mg, 3.90 mmol, 2 eq) in one portion at 15° C. under N₂. The mixture wasstirred at 65° C. for 15 hours. The aqueous phase was extracted withethyl acetate (30 mL×3). The combined organic phase was washed withbrine (5 mL), dried with anhydrous Na₂SO₄, filtered and concentrated invacuum. Compound 18.2 (50 mg, 155.56 μmol, 7.98% yield) was obtained ascolorless oil.

A mixture of compound 18.2 (50 mg, 155.56 μmol, 1 eq) in HCl/EtOAc (4 M,10 mL, 257.14 eq), the mixture was stirred at 15° C. for 15 hours. Thenthe mixture was concentrated in vacuum. Compound 18.1 (50 mg, crude) wasobtained as a white solid. LCMS (ESI): m/z: [M+H] called for C₁₂H₁₉N₃O:220; found 220; RT=0.114 min.

To a mixture of compound 18.1 (50 mg, 225.94 μmol, 1 eq) and DIEA (58.40mg, 451.88 μmol, 78.92 μL, 2 eq) in DMF (2 mL) was added carbononitridicbromide (23.93 mg, 225.94 μmol, 16.62 μL, 1 eq) in one portion at 0° C.under N₂. The mixture was stirred at 15° C. for 15 hours. The aqueousphase was extracted with ethyl acetate (30 mL×3). The combined organicphase was washed with brine (5 mL), dried with anhydrous Na₂SO₄,filtered and concentrated in vacuum. The residue was purified byprep-HPLC (basic condition). Product 18 (1.20 mg, 4.87 μmol, 2.16%yield) was obtained as a white solid. ¹H NMR (400 MHz, chloroform-d) δppm 1.35-1.75 (m, 5H) 1.77-2.01 (m, 6H) 2.02-2.21 (m, 2H) 3.33-3.54 (m,2H) 3.62 (br d, 7=6.15 Hz, 2H) 3.88-4.10 (m, 2H) 4.41 (br s, 2H) 5.98(s, 1H) 6.14-6.37 (m, 1H) 7.76-8.02 (m, 1H). LCMS (ESI): m/z: [M+H]called for C₁₃H₁₈N₄O: 247; found 247; RT=1.044 min.

Example 19. Preparation of(S)-2-(((2-aminopyridin-4-yl)oxy)methyl)pyrrolidine-1-carbonitrile (19)

To a mixture of compound 19.4 (1 g, 4.97 mmol, 1 eq) and4-methylbenzenesulfonyl chloride (1.14 g, 5.96 mmol, 1.20 eq) in DCM (15mL) was added DMAP (97.12 mg, 794.99 μmol, 0.16 eq) and TEA (754.17 mg,7.45 mmol, 1.03 mL, 1.50 eq) in one portion at 0° C. under N₂. Themixture was then heated to 18° C. and stirred for 10 hours. The reactionmixture was diluted with H₂O 20 mL and extracted with EtOAc 45 mL (15mL×3). The combined organic layers were washed with brine 20 mL (20mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give compound 19.3 (1.70 g, crude) as a colorless oil. LCMS(ESI): m/z: [M+H] calcd for C₁₇H₂₅NSO₅: 355; found 300; RT=0.890 min.

To a solution of compound 19.5 (579.28 mg, 5.26 mmol, 1.10 eq) in DMF(20 mL) was added NaH (172.18 mg, 7.17 mmol, 1.50 eq) portionwise at 25°C. under N₂. The mixture was stirred at 25° C. for 30 mins, then wasadded compound 19.3 (1.70 g, 4.78 mmol, 1 eq). The mixture was heated to60° C. and stirred for 9.5 hours. The reaction mixture was diluted withH₂O 20 mL and extracted with EtOAc 45 mL (15 mL×3). The combined organiclayers were washed with brine 20 mL (20 mL×1), dried over Na₂SO₄,filtered and concentrated under reduced pressure to give a residue. Theresidue was purified by column chromatography (SiO₂, DCM:MeOH=20:1) togive compound 19.2 (700 mg, 2.39 mmol, 49.92% yield) as a colorless oil.LCMS (ESI): m/z: [M+H] calcd for C₁₅H₂₃N₃O₃: 293; found 294; RT=0.667min.

Compound 19.2 (700 mg, 2.39 mmol, 1 eq) was added into a solution ofHCl/MeOH (10 mL). The mixture was stirred at 18° C. for 15 hours. Thereaction mixture was concentrated under reduced pressure to givecompound 19.1 (550 mg, crude) as a yellow solid. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 1.74 (dq, 0.7=12.66, 8.20 Hz, 1H) 1.83-2.05 (m, 2H)2.06-2.18 (m, 1H) 3.14-3.28 (m, 2H) 3.67 (br s, 1H) 3.87-3.99 (m, 1H)4.40 (d, J=5.90 Hz, 2H) 6.43 (d, 0.7=2.51 Hz, 1H) 6.54 (dd, J=7.28, 2.51Hz, 1H) 7.92 (d, J=7.28 Hz, 1H) 8.04 (br s, 2H).

To a mixture of compound 19.1 (200 mg, 1.03 mmol, 1 eq) and DIEA (532.47mg, 4.12 mmol, 719.55 μL, 4 eq) in DCM (3 mL) was added carbononitridicbromide (109.10 mg, 1.03 mmol, 75.76 μL, 1 eq) in one portion at 0° C.under N2. The mixture was stirred at 0° C. for 30 mins. The reactionmixture diluted with H2O 3 mL and extracted with DCM 9 mL (3 mL×3). Thecombined organic layers were washed with brine 4 mL (4 mL×1), dried overNa2SO4, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by prep-HPLC (neutral condition) togive product 19 (30 mg, 137.46 μmol, 13.35% yield) as a yellow solid.LCMS (ESI): m/z: [M+H] calcd for C₁₁H₁₄N₄O: 218; found 219; RT=1.953min. ¹H NMR (400 MHz, DMSO-d) δ ppm 1.72 (dq, J=12.27, 6.16 Hz, 1H)1.82-1.96 (m, 2H) 1.99-2.09 (m, 1H) 3.36-3.49 (m, 2H) 3.89-4 (m, 2H)4-4.07 (m, 1H) 5.81 (s, 2H) 5.97 (d, J=2.13 Hz, 1H) 6.14 (dd, J=5.77,2.26 Hz, 1H) 7.74 (d, J=5.77 Hz, 1H).

Example 20. Preparation of(R)-2-(((2-aminopyridin-4-yl)oxy)methyl)pyrrolidine-1-carbonitrile (20)

To a mixture of compound 20.4 (1 g, 4.97 mmol, 1 eq) and4-methylbenzenesulfonyl chloride (1.14 g, 5.96 mmol, 1.20 eq) in DCM (15mL) was added DMAP (97.12 mg, 794.99 μmol, 0.16 eq) and TEA (754.17 mg,7.45 mmol, 1.03 mL, 1.50 eq) in one portion at 0° C. under N₂. Themixture was then heated to 25° C. and stirred for 10 hours. The reactionmixture was diluted with H₂O 20 mL and extracted with EtOAc 15 mL (15mL×3). The combined organic layers were washed with brine 20 mL (20mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give compound 20.3 (1.90 g, crude) as a yellow oil. ¹H NMR(400 MHz, chloroform-d) ppm 1.30-1.48 (m, 9H) 1.71-2.02 (m, 4H)2.39-2.51 (m, 3H) 3.21-3.42 (m, 2H) 3.80-4.03 (m, 1H) 4.09 (d, J=6.15Hz, 1H) 7.35 (d, J=4.77 Hz, 2H) 7.78 (d, J=8.16 Hz, 2H). LCMS (ESI):m/z: [M+H] calcd for C₁₇H₂₅NSO₅: 355; found 256,300; RT=1.119 min.

To a solution of compound 20.5 (647.43 mg, 5.88 mmol, 1.10 eq) in DMF(20 mL) was added NaH (192.43 mg, 8.02 mmol, 1.50 eq) portionwise at 0°C. under N₂. The mixture was stirred at 0° C. for 30 mins, then wasadded compound 20.3 (1.90 g, 5.35 mmol, 1 eq). The mixture was heated to60° C. and stirred for 9.5 hours. The reaction mixture was diluted withH₂O 20 mL and extracted with EtOAc 15 mL (15 mL×3). The combined organiclayers were washed with brine 20 mL (20 mL×1), dried over Na₂SO₄,filtered and concentrated under reduced pressure to give a residue. Thenthe residue was purified by column (SiO2, DCM/MeOH=10:1) to givecompound 20.2 (900 mg, 3.07 mmol, 57.34% yield) as a yellow oil. ¹H NMR(400 MHz, methanol-d4) ppm 1.82-1.93 (m, 1H) 1.95-2.08 (m, 3H) 3.36-3.41(m, 2H) 3.90-4.02 (m, 1H) 4.04-4.20 (m, 2H) 6.12 (d, J=15.94 Hz, 1H)6.26 (dd, J=6.09, 2.32 Hz, 1H) 7.64-7.76 (m, 1H). LCMS (ESI): m/z: [M+H]calcd for C₁₅H₂₃N₃O₃: 293; found 294; RT=0.671 min.

Compound 20.2 (900 mg, 3.07 mmol, 1 eq) was added into a solution ofHCl/MeOH (10 mL). The mixture was stirred at 18° C. for 15 hours. Thereaction mixture was concentrated under reduced pressure to givecompound 20.1 (900 mg, crude) as a yellow solid. ¹H NMR (400 MHz,methanol-d4) ppm 1.84-1.96 (m, 1H) 1.98-2.20 (m, 2H) 2.21-2.35 (m, 1H)3.27 (dt, J=3.20, 1.71 Hz, 1H) 3.33-3.41 (m, 2H) 4.28-4.41 (m, 1H) 4.49(dd, J=11.03, 3.53 Hz, 1H) 6.46 (d, J=2.21 Hz, 1H) 6.58 (dd, J=7.28,2.43 Hz, 1H) 7.70-7.82 (m, 1H). LCMS (ESI): m/z: [M+H] calcd forC₁₀H₁₅N₃O: 193; found 194; RT=0.086 min.

To a mixture of compound 20.1 (200 mg, 1.03 mmol, 1 eq) and DIEA (532.47mg, 4.12 mmol, 719.55 μL, 4 eq) in DCM (3 mL) was added carbononitridicbromide (109.10 mg, 1.03 mmol, 75.76 μL, 1 eq) in one portion at 0° C.under N₂. The mixture was stirred at 0° C. for 30 mins. The reactionmixture diluted with H₂O 3 mL and extracted with DCM 3 mL (3 mL×3). Thecombined organic layers were washed with brine 4 mL (4 mL×1), dried overNa₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by prep-HPLC (neutral condition) togive product 20 (100 mg, 458.19 μmol, 44.48% yield) as a yellow solid.¹H NMR (400 MHz, DMSO-d6) δ ppm 1.71 (dq, J=12.23, 6.17 Hz, 1H)1.79-1.96 (m, 2H) 1.97-2.11 (m, 1H) 3.35-3.49 (m, 2H) 3.89-4 (m, 2H)4-4.08 (m, 1H) 5.93 (s, 1H) 5.99 (d, J=2.26 Hz, 1H) 6.11-6.20 (m, 1H)7.74 (d, J=6.02 Hz, 1H). LCMS (ESI): m/z: [M+H] calcd for C₁₁H₁₄N₄O:218; found 219; RT=1.981 min.

Example 21. Preparation of2-(2-(2-aminopyridin-4-yl)ethyl)pyrrolidine-1-carbonitrile (21)

To a solution of compound 21.12 (58 g, 419.92 mmol, 1 eq) in MeOH (600mL) was added SOCl₂ (99.92 g, 839.84 mmol, 60.93 mL, 2 eq) dropwise at0° C. under N₂. The mixture was stirred at 0° C. for 30 mins, then washeated to 18° C. and stirred at 18° C. for 14.5 hours. The reactionmixture was concentrated under reduced pressure to give compound 21.11(65 g, crude) as a white solid. LCMS (ESI): m/z: [M+H] calcd forC₁H₈N₂O₂: 152; found 153; RT=0.100 min.

To a mixture of compound 21.11 (65 g, 427.21 mmol, 1 eq) and DMAP (2.61g, 21.36 mmol, 0.05 eq) in t-BuOH (500 mL) and acetone (150 mL) wasadded Boc₂O (279.72 g, 1.28 mol, 294.44 mL, 3 eq) dropwise at 18° C.under N₂. The mixture was stirred at 18° C. for 15 hours. The solutionwas diluted with pentane (200 ml), cooled in the refrigerator for 3hours and filtered to obtain compound 21.10 (110 g, 312.16 mmol, 73.07%yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C₁₇H₂₄N₂O₆:352; found 353; RT=0.877 min. ¹H NMR (400 MHz, chloroform-d) δ ppm1.39-1.50 (m, 19H) 3.97 (s, 3H) 7.77 (dd, J=5.02, 1.38 Hz, 1H) 7.82 (s,1H) 8.62 (d, J=5.02 Hz, 1H)

To a solution of compound 21.10 (60 g, 170.27 mmol, 1 eq) in THF (1 L)was added LiAlH4 (12.92 g, 340.54 mmol, 2 eq) portionwise at 0° C. underN₂. The mixture was stirred at 0° C. for 1 hours, then heated to 18° C.and stirred at 18° C. for 14 hours. The reaction mixture was quenched byaddition 8% NaOH (15 ml), filtered and then diluted with H₂O 1000 mL andextracted with EtOAc 1500 mL (500 mL×3). The combined organic layerswere washed with brine 1000 mL (1000 mL×1), dried over Na₂SO₄, filteredand concentrated under reduced pressure to give a residue. The residuewas purified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=2:1) to give compound 21.9 (15 g, 66.89 mmol, 39.28% yield) as awhite solid. LCMS (ESI): m/z: [M+H] calcd for C₁₁H₁₆N₂O₃: 293; found294; RT=0.313 min.

To a solution of compound 21.9 (8 g, 35.67 mmol, 1 eq) in DCM (60 mL)was added Dess-Martin periodinane (18.16 g, 42.81 mmol, 13.25 mL, 1.20eq) portionwise at 18° C. under N₂. The mixture was stirred at 18° C.for 2 hours. The reaction mixture was diluted with H₂O 60 mL andextracted with DCM 150 mL (50 mL×3). The combined organic layers werewashed with brine 100 mL (100 mL×1), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=5:1) to give compound 21.8 (5.10 g, 22.95 mmol, 64.33% yield) asa white solid. LCMS (ESI): m/z: [M+H] calcd for C₁₁H₁₄N₂O₃: 222; found223; RT=0.313 min. ¹H NMR (400 MHz, chloroform-d) δ ppm 1.59 (s, 9H)7.41 (dd, J=5.08, 1.32 Hz, 1H) 8.48 (s, 1H) 8.52 (d, J=5.15 Hz, 1H) 8.83(br s, 1H).

To a mixture of compound 21.8 (2.50 g, 11.25 mmol, 1 eq) and methyl2-dimethoxyphosphorylacetate 21.7 (2.05 g, 11.25 mmol, 1.63 mL, 1 eq) inTHF (30 mL) was added K₂CO₃ (3.11 g, 22.50 mmol, 2 eq) in one portion at50° C. under N₂. The mixture was stirred at 50° C. for 15 hours. Thereaction mixture was diluted with H₂O 30 mL and extracted with EtOAc 90mL (30 mL×3). The combined organic layers were washed with brine 50 mL(50 mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=5:1) to givecompound 21.6 (2.40 g, 8.62 mmol, 76.62% yield) as a white solid.

LCMS (ESI): m/z: [M+H] calcd for C₁₄H₁₈N₂O₄: 278; found 279; RT=0.739min.

To a solution of compound 21.6 (1.40 g, 5.03 mmol, 1 eq) in MeOH (100mL) was added Pd—C (10%, 0.2 g) under N₂. The suspension was degassedunder vacuum and purged with H₂ several times. The mixture was stirredunder H₂ (50 psi) at 20° C. for 15 hours. The reaction mixture wasfiltered and concentrated under reduced pressure to give a residue. Theresidue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=3:1) to give compound 21.5 (1.30 g, 4.64 mmol,92.20% yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd forC₁₄H₂₀N₂O₄: 280; found 281; RT=0.617 min. ¹H NMR (400 MHz, chloroform-d)δ ppm 1.47 (s, 9H) 2.59 (t, J=7.78 Hz, 2H) 2.78-2.98 (m, 2H) 3.50-3.71(m, 3H) 6.66-6.82 (m, 1H) 7.79 (s, 1H) 8.13 (d, J=5.15 Hz, 1H) 8.83 (s,1H)

To a solution of DIPA (505.95 mg, 5 mmol, 702.71 μL, 2 eq) in THF (10mL) was added n-BuLi (2.5 M, 1.50 mL, 1.50 eq) dropwise at −78° C. underN₂. The mixture was then added compound 21.4 (589.84 mg, 3.75 mmol,601.88 μL, 1.50 eq) in one portion at −78° C., the mixture was stirredat −78° C. for 30 mins, then was added compound 21.5 (700 mg, 2.50 mmol,1 eq) in one portion at −78° C., the mixture was heated to 18° C. andstirred for 14.5 hours. The reaction mixture was quenched by additionH₂O 10 mL and then diluted with EtOAc 5 mL and extracted with EtOAc 15mL (5 mL×3). The combined organic layers were washed with brine 10 mL(10 mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by prep-TLC (SiO₂,PE:EA=2:1) to give compound 21.3 (180 mg, 539.92 μmol, 21.60% yield) asa white solid. LCMS (ESI): m/z: [M+H] calcd for C₁₇H₂₃N₃O₄: 333; found334; RT=0.597 min.

Compound 21.3 (180 mg, 539.92 μmol, 1 eq) was mixed with 6 N HCl (5 mL),heated at 100° C. for 15 hours. The mixture was concentrated at reducedpressure to a syrup and then basified with 10% KOH (10 ml), theresulting two phase mixture was extracted with EtOAc 15 ml (5 ml×3) andthe extract dry Na₂SO₄ and concentrated to give compound 21.2 (90 mg,crude) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C₁₆H₂₃N₃O₂:189; found 190; RT=0.097 min.

To a mixture of compound 21.2 (90 mg, 475.54 μmol, 1 eq) and NaBH₃CN(44.82 mg, 713.30 μmol, 1.50 eq) in MeOH (1 mL) was added HCl/MeOH (500μL) in one portion at 18° C. under N₂. The mixture was stirred at 18° C.for 4 hours. The reaction mixture was concentrated under reducedpressure to give a residue, then was diluted with H₂O 2 mL and extractedwith EtOAc 6 mL (2 mL×3). The combined organic layers were washed withbrine 5 mL (5 mL×1), dried over Na₂SO₄, filtered and concentrated underreduced pressure to give compound 21.1 (90 mg, 470.54 μmol, 98.95%yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C₁₁H₁₇N₃: 191;found 192; RT=0.095 min. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.27-1.39 (m,2H) 1.64-1.85 (m, 4H) 1.88-1.99 (m, 1H) 2.43-2.59 (m, 2H) 2.78-2.88 (m,1H) 2.93-3.04 (m, 2H) 5.86 (s, 2H) 6.38 (s, 1H) 6.46 (dd, 7=5.29, 1.32Hz, 1H) 7.89 (d, 7=5.07 Hz, 1H).

To a mixture of compound 21.1 (40 mg, 209.13 μmol, 1 eq) and DIEA(108.11 mg, 836.51 μmol, 146.10 μL, 4 eq) in DMF (1 mL) was addedcarbononitridic bromide (22.15 mg, 209.13 μmol, 15.38 μL, 1 eq) in oneportion at 0° C. under N₂. The mixture was stirred at 0° C. for 30 mins.The residue was purified by prep-HPLC (neutral condition) to giveproduct 21 (5 mg, 23.12 μmol, 11.05% yield) as a white solid. LCMS(ESI): m/z: [M+H] calcd for C₁₂H₁₆N₄: 216; found 217; RT=2.153 min. ¹HNMR (400 MHz, DMSO-d₆) δ ppm 1.48-1.60 (m, 1H) 1.60-1.70 (m, 1H)1.76-1.93 (m, 3H) 1.94-2.04 (m, 1H) 2.42-2.47 (m, 2H) 3.35-3.44 (m, 2H)3.48-3.57 (m, 1H) 5.77 (s, 2H) 6.28 (s, 1H) 6.33-6.40 (m, 1H) 7.78 (d,7=5.15 Hz, 1H).

Example 22. Preparation of2-(((2-(methylamino)pyridin-4-yl)oxy)methyl)pyrrolidine-1-carbonitrile(22)

To a mixture of compound 22.6 (4.50 g, 22.36 mmol, 1 eq) and4-methylbenzenesulfonyl chloride (5.12 g, 26.83 mmol, 1.20 eq) in DCM(15 mL) was added DMAP (437.06 mg, 3.58 mmol, 0.16 eq) and TEA (3.39 g,33.54 mmol, 4.65 mL, 1.50 eq) in one portion at 0° C. under N₂. Themixture was then heated to 25° C. and stirred for 10 hours. The reactionmixture was diluted with H₂O 20 mL and extracted with EtOAc 15 mL (15mL×3). The combined organic layers were washed with brine 20 mL (20mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give compound 22.5 (8 g, crude) as a colorless oil. ¹H NMR(400 MHz, chloroform-d) δ ppm 1.20-1.52 (m, 9H) 1.81 (br. s., 1H) 1.93(br. s., 2H) 2.39-2.54 (m, 3H) 3.17-3.44 (m, 2H) 3.80-4.04 (m, 1H) 4.10(d, J=6.02 Hz, 1H) 7.30-7.42 (m, 2H) 7.67-7.86 (m, 2H).

To a solution of compound 22.7 (2.73 g, 24.76 mmol, 1.10 eq) in DMF (20mL) was added NaH (810.24 mg, 33.76 mmol, 1.50 eq) portionwise at 0° C.under N₂. The mixture was stirred at 0° C. for 30 mins, then was addedcompound 22.5 (8 g, 22.51 mmol, 1 eq). The mixture was heated to 60° C.and stirred for 9.5 hours. The reaction mixture was diluted with H₂O 20mL and extracted with EA 15 mL (15 mL×3). The combined organic layerswere washed with brine 20 mL (20 mL×1), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, DCM:MeOH=20:1) to give compound22.4 (4 g, 13.64 mmol, 60.57% yield) as a yellow oil. ¹H NMR (400 MHz,chloroform-d) δ ppm 1.40-1.57 (m, 9H) 1.78-2.05 (m, 4H) 3.37 (d, J=19.07Hz, 2H) 3.66-4 (m, 1H) 4.12 (dd, J=14.05, 7.03 Hz, 2H) 4.38 (br. s., 2H)5.89-6.15 (m, 1H) 6.28 (d, J=4.02 Hz, 1H) 7.88 (br. s., 1H). LCMS (ESI):m/z: [M+H] calcd for C₁₅H₂₃N₃O₃: 293; found 294; RT=0.681 min.

To a mixture of compound 22.4 (800 mg, 2.73 mmol, 1 eq) and TEA (828.75mg, 8.19 mmol, 1.14 mL, 3 eq) in THF (10 mL) was added Boc₂O (714.99 mg,3.28 mmol, 752.62 μL, 1.20 eq) in one portion at 18° C. under N₂. Themixture was stirred at 18° C. for 10 hours. The reaction mixture wasdiluted with H₂O 10 mL and extracted with EtOAc 10 mL (10 mL×3). Thecombined organic layers were washed with brine 20 mL (20 mL×1), driedover Na₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by prep-TLC (SiO₂, DCM:MeOH=10:1) togive compound 22.3 (400 mg, 1.02 mmol, 37.24% yield) as a white solid.¹H NMR (400 MHz, chloroform-d) δ ppm 1.47 (s, 9H) 1.53 (s, 9H) 1.86 (br.s., 1H) 2 (br. s., 3H) 3.21-3.55 (m, 2H) 3.83-4.05 (m, 1H) 4.13 (q,J=7.03 Hz, 4H) 6.55 (br. s., 1H) 7.52 (br. s., 1H) 7.72-7.92 (m, 1H)8.05 (d, J=6.02 Hz, 1H). LCMS (ESI): m/z: [M+H] calcd for C₂₀H₃₁N₃O₅:393; found 394; RT=0.764 min.

To a solution of compound 22.3 (210 mg, 533.70 μmol, 1 eq) in THF (5 mL)was added NaH (21.35 mg, 533.70 μmol, 60% purity, 1 eq) portionwise at0° C. under N₂. The mixture was stirred at 0° C. for 30 mins, then themixture was added CH₃I (75.75 mg, 533.70 μmol, 33.22 μL, 1 eq) dropwiseat 0° C., the mixture was heated to 18° C. and stirred for 1.5 hours.The reaction mixture was diluted with H₂O 2 mL and extracted with EtOAc2 mL (2 mL×3). The combined organic layers were washed with brine 5 mL(5 mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by prep-TLC (SiO₂,DCM:MeOH=10:1) to give compound 22.2 (160 mg, 392.64 μmol, 73.57% yield)as a colorless oil, Combined ET6889-326 and ET6889-329 to affordcompound 10 (210 mg). LCMS (ESI): m/z: [M+H] calcd for C₂₁H₃₃N₃O₅: 407;found 408; RT=0.777, 0.813 min.

Compound 22.2 (210 mg, 515.34 μmol, 1 eq) was added into a solution ofHCl/EtOAc (5 mL). The mixture was stirred at 18° C. for 15 hours. Thereaction mixture was concentrated under reduced pressure to givecompound 22.1 (200 mg, crude) as a yellow solid. LCMS (ESI): m/z: [M+H]calcd for C₁₁H₁₇N₃O: 207; found 208; RT=0.093, 0.191 min.

To a mixture of compound 22.1 (100 mg, 356.90 μmol, 1 eq, 2HCl) and DIEA(184.50 mg, 1.43 mmol, 249.32 μL, 4 eq) in DMF (2 mL) was addedcarbononitridic bromide (30.24 mg, 285.52 μmol, 21 μL, 0.80 eq) in oneportion at 0° C. under N₂. The mixture was stirred at 0° C. for 30 mins.The reaction mixture diluted with H₂O 3 mL and extracted with DCM 3 mL(3 mL×3). The combined organic layers were washed with brine 4 mL (4mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by prep-HPLC(neutral condition) to give product 22 (5 mg, 21.53 μmol, 6.03% yield)as a yellow solid. ¹H NMR (400 MHz, chloroform-d) δ ppm 1.85-2.07 (m,3H) 2.08-2.20 (m, 1H) 2.90 (d, J=5.27 Hz, 3H) 3.40-3.51 (m, 1H)3.51-3.60 (m, 1H) 3.93-4.02 (m, 1H) 4.02-4.09 (m, 2H) 5.78-5.93 (m, 1H)6.20 (dd, J=5.84, 2.07 Hz, 1H) 7.87-8 (m, 1H). LCMS (ESI): m/z: [M+H]calcd for C₁₂H₁₆N₄O: 232; found 233; RT=0.976 min.

Example 23. Preparation of2-(((2-(dimethylamino)pyridin-4-yl)oxy)methyl)pyrrolidine-1-carbonitrile(23)

To a mixture of compound 23.5 (4.50 g, 22.36 mmol, 1 eq) and4-methylbenzenesulfonyl chloride (5.12 g, 26.83 mmol, 1.20 eq) in DCM(15 mL) was added DMAP (437.06 mg, 3.58 mmol, 0.16 eq) and TEA (3.39 g,33.54 mmol, 4.65 mL, 1.50 eq) in one portion at 0° C. under N₂. Themixture was then heated to 25° C. and stirred for 10 hours. The reactionmixture was diluted with H₂O 20 mL and extracted with EtOAc 45 mL (15mL×3). The combined organic layers were washed with brine 20 mL (20mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give compound 23.4 (8 g, crude) as a colorless oil. ¹H NMR(400 MHz, chloroform-d) δ ppm 1.20-1.52 (m, 9H) 1.81 (br. s., 1H) 1.93(br. s., 2H) 2.39-2.54 (m, 3H) 3.17-3.44 (m, 2H) 3.80-4.04 (m, 1H) 4.10(d, J=6.02 Hz, 1H) 7.30-7.42 (m, 2H) 7.67-7.86 (m, 2H).

To a solution of compound 23.4 (2.73 g, 24.76 mmol, 1.10 eq) in DMF (20mL) was added NaH (810.24 mg, 33.76 mmol, 1.50 eq) portionwise at 0° C.under N₂. The mixture was stirred at 0° C. for 30 mins, then was addedcompound 23.6 (8 g, 22.51 mmol, 1 eq). The mixture was heated to 60° C.and stirred for 9.5 hours. The reaction mixture was diluted with H₂O 20mL and extracted with EtOAc 45 mL (15 mL×3). The combined organic layerswere washed with brine 20 mL (20 mL×1), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, DCM:MeOH=20:1) to give compound23.3 (4 g, 13.64 mmol, 60.57% yield) as a yellow oil. ¹H NMR (400 MHz,chloroform-d) δ ppm 1.40-1.57 (m, 9H) 1.78-2.05 (m, 4H) 3.37 (d, J=19.07Hz, 2H) 3.66-4 (m, 1H) 4.12 (dd, J=14.05, 7.03 Hz, 2H) 4.38 (br. s., 2H)5.89-6.15 (m, 1H) 6.28 (d, J=4.02 Hz, 1H) 7.88 (br. s., 1H). LCMS (ESI):m/z: [M+H] calcd for C₁₅H₂₃N₃O₃: 293; found 294; RT=0.681 min.

To a solution of compound 23.3 (100 mg, 340.88 μmol, 1 eq) in H2O (250μL) and ACETONITRILE (1 mL) was added HCHO (327 mg, 4.03 mmol, 300 μL,37% purity, 11.82 eq) and NaBH3CN (64.26 mg, 1.02 mmol, 3 eq) in oneportion at 0° C. under N₂. The mixture was stirred at 0° C. for 30 mins,then was added CH3COOH (21 mg, 349.71 μmol, 20 μL, 1.03 eq) dropwise at0° C., then heated to 18° C. and stirred for 1.5 hours. The reactionmixture was quenched by addition H₂O 5 mL then diluted with DCM 3 mL andextracted with DCM 3 mL (3 mL×3). The combined organic layers werewashed with brine 5 mL (5 mL×1), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by prep-TLC (SiO₂, DCM:MeOH=10:1) to give compound 23.2 (60 mg,186.68 μmol, 54.76% yield) as a colorless oil. LCMS (ESI): m/z: [M+H]calcd for C₁₇H₂₇N₃O₃: 321; found 322; RT=0.712 min.

Compound 23.2 (60 mg, 186.67 μmol, 1 eq) was added into a solution ofHCl/EtOAc (3 mL). The mixture was stirred at 18° C. for 1.5 hours. Thereaction mixture was concentrated under reduced pressure to givecompound 23.1 (60 mg, crude) as a yellow solid. LCMS (ESI): m/z: [M+H]calcd for C₁₂H₁₉N₃O: 221; found 222; RT=0.096 min.

To a mixture of compound 23.1 (60 mg, 232.77 μmol, 1 eq, HCl) and DIEA(120.34 mg, 931.08 μmol, 162.62 μL, 4 eq) in DMF (2 mL) was addedcarbononitridic bromide (24.65 mg, 232.77 μmol, 17.12 μL, 1 eq) in oneportion at 0° C. under N₂. The mixture was stirred at 0° C. for 30 mins.The residue was purified by prep-HPLC (neutral condition) to giveproduct 23 (10 mg, 40.60 μmol, 17.44% yield) as a white solid. LCMS(ESI): m/z: [M+H] calcd for C₁₃H₁₈N₄O: 246; found 247; RT=2.441 min. ¹HNMR (400 MHz, chloroform-d) δ ppm 1.91-2.08 (m, 3H) 2.10-2.21 (m, 1H)3.10 (s, 6H) 3.45-3.52 (m, 1H) 3.54-3.61 (m, 1H)3.97-4.04 (m, 1H)4.04-4.11 (m, 2H) 5.99 (d, J=2.01 Hz, 1H) 6.19 (dd, J=5.77, 2.01 Hz, 1H)8.04 (d, J=5.90 Hz, 1H)

Example 24. Preparation of2-(((2-aminopyridin-4-yl)oxy)methyl)indoline-1-carbonitrile (24)

To a solution of compound 24.6 (950 mg, 3.61 mmol, 1 eq) in THF (15 mL)was added BH₃-Me₂S (10 M, 721.64 μL, 2 eq) at 0° C. The mixture wasstirred at 25° C. for 15 hours. The reaction mixture was quenched byaddition H₂O 30 mL at 25° C. and extracted with ethyl acetate (15 mL×3).The combined organic layers were washed with saturated brines (5 mL×1),dried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure to give a crude product. Compound 24.5 (750 mg, 3.01mmol, 83.34% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H]called for C₁₄H₁₉NO₃: 250; found 250; RT=0.800 min.

To a solution of compound 24.5 (750 mg, 3.01 mmol, 1 eq) in DCM (20 mL)was added TEA (1.52 g, 15.04 mmol, 2.09 mL, 5 eq) and TosCl (1.72 g,9.03 mmol, 3 eq) and DMAP (183.77 mg, 1.50 mmol, 0.50 eq). The mixturewas stirred at 25° C. for 15 hours. The reaction mixture was quenched byaddition H₂O 50 mL at 25° C. and extracted with DCM (50 mL×3). Thecombined organic layers were washed with saturated brines (15 mL×1),dried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure to give a crude product. Compound 24.4 (1.40 g, crude)was obtained as a yellow oil. LCMS (ESI): m/z: [M+H] called forC₂₁H₂₅NO₅S: 404; found 404; RT=0.925 min.

To a solution of compound 24.4 (1.40 g, 3.47 mmol, 1 eq) in DMF (30 mL)was added K2CO3 (959.10 mg, 6.94 mmol, 2 eq) and compound 24.3 (458.46mg, 4.16 mmol, 1.20 eq). The mixture was stirred at 80° C. for 15 hours.The reaction mixture was quenched by addition H₂O 50 mL at 25° C. andextracted with ethyl acetate (50 mL×3). The combined organic layers werewashed with saturated brines (15 mL×1), dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give acrude product. Compound 24.2 (200 mg, 585.82 μmol, 16.88% yield) wasobtained as a yellow oil. ¹H NMR (400 MHz, chloroform-d) δ ppm 1.59 (s,9H) 2.46 (s, 1H) 3.01-3.13 (m, 1H) 3.35 (dd, J=16.25, 9.72 Hz, 1H) 3.90(br s, 1H) 4.22 (br s, 1H) 4.38-4.61 (m, 2H) 4.79 (br s, 1H) δ (br s,1H) 6.22-6.31 (m, 1H) 6.93-7.03 (m, 1H) 7.14-7.23 (m, 2H) 7.87 (d,J=5.77 Hz, 1H). LCMS (ESI): m/z: [M+H] called for C₁₉H₂₃N₃O₃: 342; found342; RT=0.759 min.

A mixture of compound 24.2 (50 mg, 146.46 μmol, 1 eq) in HCl/EtOAc (5mL) was stirred at 25° C. for 15 hours. The mixture was concentratedunder reduced pressure to give a residue. Compound 24.1 (45 mg, 143.22μmol, 97.79% yield, 2HCl) was obtained as a yellow oil. LCMS (ESI): m/z:[M+H] called for C₁₄H₁₅N₃O: 242; found 242; RT=0.129 min.

To a solution of compound 24.1 (40 mg, 127.30 μmol, 1 eq, 2HCl) in DMSO(2 mL) was added NaHCO₃ (53.47 mg, 636.52 μmol, 24.76 μL, 5 eq) and BrCN(13.48 mg, 127.30 μmol, 9.36 μL, 1 eq). The mixture was stirred at 60°C. for 2 hours. The residue was purified by prep-HPLC (TFA condition).Product 24 (1 mg, 3.76 μmol, 2.95% yield) was obtained as a yellowsolid. ¹H NMR (400 MHz, methanol-d₄) δ ppm 3.10 (dd, J=16.44, 5.90 Hz,1H) 3.51 (dd, J=16.44, 10.04 Hz, 1H) 4.32-4.43 (m, 1H) 4.45-4.54 (m, 1H)4.78-4.84 (m, 1H) 6.39 (d, J=2.51 Hz, 1H) 6.53 (dd, J=7.28, 2.38 Hz, 1H)6.93 (d, J=8.03 Hz, 1H) 7.02-7.10 (m, 1H) 7.26 (br d, J=7.15 Hz, 2H)7.73 (d, J=7.28 Hz, 1H). LCMS (ESI): m/z: [M+H] called for C₁₅H₁₄N₄O:267; found 267; RT=1.955 min.

Example 25. Preparation of1-(((2-aminopyridin-4-yl)oxy)methyl)isoindoline-2-carbonitrile (25)

To a solution of compound 25.6 (800 mg, 3.04 mmol, 1 eq) in THF (8 mL)was added BH₃-Me₂S (10 M, 608 μL, 2 eq) at 0° C. The mixture was stirredat 25° C. for 15 hours. The reaction mixture was quenched by additionH₂O 20 mL at 25° C. and extracted with ethyl acetate (15 mL×3). Thecombined organic layers were washed with saturated brines (10 mL×1),dried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure to give a crude product. Compound 25.5 (600 mg, 2.41mmol, 79.17% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H]called for C₁₄H₁₉NO₃: 250; found 250; RT=0.752 min.

To a solution of compound 25.5 (100 mg, 401.11 μmol, 1 eq) in DCM (2 mL)was added TosCl (229.41 mg, 1.20 mmol, 3 eq) and TEA (202.94 mg, 2.01mmol, 278 μL, 5 eq) and DMAP (14.70 mg, 120.33 μmol, 0.30 eq). Themixture was stirred at 25° C. for 15 hours. The reaction mixture wasquenched by addition H₂O 10 mL at 25° C. and extracted with DCM (15mL×3). The combined organic layers were washed with saturated brines (5mL×1), dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure to give a crude product. Compound 25.4 (230 mg,crude) was obtained as a yellow oil. ¹H NMR (400 MHz, chloroform-d) δppm 1.07-1.21 (m, 1H) 1.32-1.42 (m, 2H) 1.32-1.42 (m, 1H) 1.44-1.55 (m,8H) 2.26-2.50 (m, 5H) 3.02-3.36 (m, 1H) 3.02-3.36 (m, 5H) 4.15-4.81 (m,4H) 6.64 (d, J=7.03 Hz, 1H) 7.11-7.41 (m, 8H) 7.56-7.72 (m, 2H) 7.83 (d,J=8.16 Hz, 1H) 8.24 (d, 0.7=7.15 Hz, 1H). LCMS (ESI): m/z: [M+H] calledfor C₂₁H₂₅NO₅S: 404; found 404; RT=0.925 min.

To a solution of compound 25.4 (230 mg, 570.03 μmol, 1 eq) in DMF (3 mL)was added K₂CO₃ (157.57 mg, 1.14 mmol, 2 eq) and compound 25.3 (75.32mg, 684.04 μmol, 1.20 eq). The mixture was stirred at 80° C. for 15hours. The reaction mixture was quenched by addition H₂O 10 mL at 25° C.and extracted with ethyl acetate (15 mL×3). The combined organic layerswere washed with saturated brines (5 mL×1), dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give acrude product. The residue was purified by column chromatography (SiO₂,DCM:MeOH=10:1). Compound 25.2 (50 mg, 146.46 μmol, 25.69% yield) wasobtained as a black brown oil. LCMS (ESI): m/z: [M+H] called forC₁₉H₂₃N₃O₃: 342; found 342; RT=0.742 min.

A mixture of compound 25.2 (50 mg, 146.46 μmol, 1 eq) in HCl/EtOAc (5mL) was stirred at 25° C. for 15 hours. The mixture was concentratedunder reduced pressure to give a residue. Compound 25.1 (30 mg, 95.48μmol, 65.19% yield, 2HCl) was obtained as a black brown oil. LCMS (ESI):m/z: [M+H] called for C₁₄H₁₅N₃O: 242; found 242; RT=0.099 min.

To a solution of compound 25.1 (30 mg, 95.48 μmol, 1 eq, 2HCl) in DMF (1mL) was added DIEA (49.36 mg, 381.91 μmol, 66.70 μL, 4 eq) and BrCN(10.11 mg, 95.48 μmol, 7.02 μL, 1 eq). The mixture was stirred at 0° C.for 5 min. The residue was purified by prep-HPLC (basic condition).Product 25 (5 mg, 18.78 μmol, 19.66% yield) was obtained as a whitesolid. ¹H NMR (400 MHz, methanol-d₄) δ ppm 4.25 (dd, J=10.36, 5.95 Hz,1H) 4.47 (dd, J=10.36, 3.53 Hz, 1H) 4.79-4.88 (m, 2H) 5.29-5.41 (m, 1H)6.12 (d, J=2.20 Hz, 1H) 6.25 (dd, J=6.06, 2.32 Hz, 1H) 7.30-7.49 (m, 4H)7.71 (d, J=6.17 Hz, 1H). LCMS (ESI): m/z: [M+H] called for C₁₅H₁₄N₄O:267; found 267; RT=2.440 min.

Example 26. Preparation ofN-((1-aminoisoquinolin-6-yl)methyl)-N-methylcyanamide (26)

To a solution of compound 26.7 (2 g, 8.25 mmol, 1 eq) in NMP (20 mL) wasadded NH₃.H₂O (18.20 g, 519.26 mmol, 20 mL, 62.94 eq). The mixture wasstirred at 150° C. for 15 hours. The reaction mixture was quenched byaddition H₂O 50 mL at 25° C. and extracted with ethyl acetate (50 mL×3).The combined organic layers were washed with saturated brines (10 mL×1),dried over anhydrous sodium sulfate, filtered and concentrated underreduced

pressure to give a crude product. Compound 26.6 (5 g, crude) wasobtained as a yellow oil. LCMS (ESI): m/z: [M+H] called for C₉H₇N₂Br:224; found 224; RT=0.589 min.

To a solution of compound 26.6 (2.50 g, 11.21 mmol, 1 eq) in THF (40 mL)was added TEA (4.54 g, 44.83 mmol, 6.21 mL, 4 eq) and Boc₂O (6.11 g,28.02 mmol, 6.44 mL, 2.50 eq) and DMAP (410.76 mg, 3.36 mmol, 0.30 eq).The mixture was stirred at 25° C. for 15 hours. The reaction mixture wasquenched by addition H₂O 50 mL at 25° C. and extracted with ethylacetate (50 mL×3). The combined organic layers were washed withsaturated brines (20 mL×1), dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a crudeproduct. The residue was purified by re-crystallization from ethylacetate (5 mL) to give compound 26.5 (3 g, 7.09 mmol, 63.22% yield) asyellow solid. ¹H NMR (400 MHz, chloroform-d) δ ppm 1.19-1.45 (m, 19H)7.57 (d, J=5.77 Hz, 1H) 7.71 (dd, J=8.97, 1.82 Hz, 1H) 7.84 (d, 0.7=8.91Hz, 1H) 8.06 (d, 0.7=1.76 Hz, 1H) 8.46 (d, J=5.77 Hz, 1H). LCMS (ESI):m/z: [M+H] called for C₁₉H₂₃BrN₂O₄: 424; found 424; RT=0.896 min.

To a solution of compound 26.5 (1.65 g, 3.90 mmol, 1 eq) in toluene (120mL) and MeOH (60 mL) was added TEA (1.58 g, 15.60 mmol, 2.16 mL, 4 eq)and Pd(dppf)Cl₂.CH₂Cl₂ (318.32 mg, 390 μmol, 0.10 eq) under N₂. Thesuspension was degassed under vacuum and purged with CO several times.The mixture was stirred under CO (50 psi) at 50° C. for 15 hours. Themixture was filtered and concentrated under reduced pressure to give aresidue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1:1). Compound 26.4 (1.20 g, 2.98 mmol,76.46% yield) was obtained as a white solid. ¹H NMR (400 MHz,methanol-d₄) δ ppm 1.07-1.58 (m, 20H) 4.01 (s, 3H) 7.99-8.04 (m, 1H)8.05-8.10 (m, 1H) 8.29 (dd, J=8.82, 1.54 Hz, 1H) 8.47 (d, J=5.73 Hz, 1H)8.74 (d, J=1.32 Hz, 1H). LCMS (ESI): m/z: [M+H] called for C₂₁H₂₆N₂O₆:403; found 403; RT=1.360 min.

A mixture of compound 26.4 (100 mg, 248.48 μmol, 1 eq) in HCl/EtOAc (20mL) was stirred at 25° C. for 15 hours. The mixture was filtered andconcentrated under reduced pressure to give a residue. Compound 26.3 (50mg, 209.49 μmol, 84.31% yield, HCl) was obtained as a yellow solid. LCMS(ESI): m/z: [M+H] called for C₁₁H₁₀N₂O₂: 203; found 203; RT=0.119 min. Amixture of compound 26.3 (50 mg, 247.27 μmol, 1 eq) in MeNH₂ (10 mL) wasstirred at 80° C. for 15 hours. The mixture was filtered andconcentrated under reduced pressure to give compound 26.2 (65 mg, crude)as a brown oil. LCMS (ESI): m/z: [M+H] called for C₁₁H₁₁N₃O: 202; found202; RT=1.093 min.

To a solution of compound 26.2 (65 mg, 323.01 μmol, 1 eq) in THF (5 mL)was added BH₃-Me₂S (10 M, 323.01 μL, 10 eq) at 0° C. The mixture wasstirred at 70° C. for 4 hours. The reaction mixture was quenched byaddition MeOH 20 mL at 25° C., then concentrated under reduced pressureto give a crude product, to the crude product was added HCl (15 mL), themixture was stirred at 25° C. for 2 hours and extracted with DCM (25mL×5). The combined organic layers were washed with saturated brines (5mL×1), dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure to give a crude product. The residue was purifiedby prep-HPLC (neutral condition). Compound 26.1 (40 mg, 213.63 μmol,66.14% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H]called for C₁₁H₁₃N₃: 188; found 188; RT=0.506 min.

To a solution of compound 26.1 (10 mg, 53.41 μmol, 1 eq) in DMF (1 mL)was added DIEA (20.71 mg, 160.23 μmol, 27.98 μL, 3 eq) and BrCN (5.66mg, 53.41 μmol, 3.93 μL, 1 eq). The mixture was stirred at 0° C. for 10min. The residue was purified by prep-HPLC (neutral condition). Product26 (1 mg, 4.71 μmol, 8.82% yield) was obtained as a yellow oil. ¹H NMR(400 MHz, chloroform-d) δ ppm 2.85 (s, 3H) 4.33 (s, 2H) 7.06 (d, J=5.90Hz, 1H) 7.52 (d, J=8.41 Hz, 1H) 7.65 (s, 1H) 7.88 (d, J=8.41 Hz, 1H)7.97 (d, J=6.02 Hz, 1H). LCMS (ESI): m/z: [M+H] called for C₁₂H₁₂N₄:213; found 213; RT=2.098 min.

Example 27. Preparation ofN-(2-((2-aminopyridin-4-yl)oxy)phenyl)-N-methylcyanamide (27)

To a solution of compound 27.6 (3 g, 27.25 mmol, 1 eq) in DMF (100 mL)was added K₂CO₃ (7.53 g, 54.50 mmol, 2 eq) and compound 27.5 (4.61 g,32.70 mmol, 3.44 mL, 1.20 eq). The mixture was stirred at 50° C. for 15hours. The reaction mixture was quenched by addition H₂O 100 mL at 25°C. and extracted with ethyl acetate (100 mL×3). The combined organiclayers were washed with saturated brines (20 mL×1), dried over anhydroussodium sulfate, filtered and concentrated under reduced pressure to givea crude product. The residue was purified by column chromatography(SiO₂, Petroleum ether/Ethyl acetate=1:1). Compound 27.4 (3.30 g, 14.27mmol, 52.38% yield) was obtained as a brown solid. ¹H NMR (400 MHz,chloroform-d) δ ppm 6.06 (d, J=1.98 Hz, 1H) 6.32 (dd, J=5.84, 2.09 Hz,1H) 7.22-7.35 (m, 1H) 7.42 (td, J=7.83, 1.10 Hz, 1H) 7.69 (ddd, J=8.21,7.44, 1.76 Hz, 1H) 7.99-8.16 (m, 2H). LCMS (ESI): m/z: [M+H] called forC₁₁H₉N₃O₃: 232; found 232; RT=0.522 min.

To a solution of compound 27.4 (3.30 g, 14.27 mmol, 1 eq) in THF (50 mL)was added TEA (5.78 g, 57.08 mmol, 7.92 mL, 4 eq) and Boc₂O (7.79 g,35.67 mmol, 8.20 mL, 2.50 eq) and DMAP (523.11 mg, 4.28 mmol, 0.30 eq).The mixture was stirred at 25° C. for 15 hours. The reaction mixture wasquenched by addition H₂O 100 mL at 25° C. and extracted with ethylacetate (50 mL×3). The combined organic layers were washed withsaturated brines (10 mL×1), dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a crudeproduct. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1). Compound 27.3 (3.20 g, 7.42 mmol,51.98% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H]called for C₂₁H₂₅N₃O₇: 432; found 432; RT=0.904 min.

To a solution of compound 27.3 (1.50 g, 3.48 mmol, 1 eq) in MeOH (150mL) was added Pd—C (10%, 150 mg) and formaldehyde (12 M, 261 μL, 0.90eq) under N₂. The suspension was degassed under vacuum and purged withH₂ several times. The mixture was stirred under H₂ (40 psi) at 40° C.for 3 hours. The reaction mixture was filtered and the filter wasconcentrated. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1). Compound 27.2 (900 mg, 2.17 mmol,62.25% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H]called for C₂₂H₂₉N₃O₅: 416; found 416; RT=0.926 min.

To a solution of compound 27.2 (100 mg, 240.69 μmol, 1 eq) in THF (2 mL)was added BrCN (43.34 mg, 409.17 μmol, 30.10 μL, 1.70 eq) and NaHCO₃(60.66 mg, 722.07 μmol, 28.08 μL, 3 eq). The mixture was stirred at 60°C. for 3 hours. The residue was purified by prep-TLC (SiO₂, Petroleumether/Ethyl acetate=2:1). Compound 27.1 (35 mg, 79.46 μmol, 33.01%yield) was obtained as a yellow oil. It was combined with a second batchfor a total of 55 mg. ¹H NMR (400 MHz, chloroform-d) δ ppm 1.46 (s, 18H)3.29 (s, 3H) 6.75 (dd, J=5.71, 2.32 Hz, 1H) 6.86 (d, J=2.26 Hz, 1H) 7.12(dd, J=7.59, 1.95 Hz, 1H) 7.30 (td, J=5.21, 2.51 Hz, 2H) 7.37-7.41 (m,1H) 8.36 (d, J=5.65 Hz, 1H). LCMS (ESI): m/z: [M+H] called forC₂₃H₂₈N₄O₅: 441; found 441; RT=0.878 min.

A mixture of compound 27.1 (55 mg, 124.86 μmol, 1 eq) in DCM (1 mL) andTFA (200 μL) was stirred at 25° C. for 15 hours. The reaction mixturewas concentrated under reduced pressure to give a residue. The residuewas purified by prep-HPLC (neutral condition). Product 27 (2 mg, 8.32μmol, 6.67% yield) was obtained as a white solid. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 3.23-3.25 (m, 3H) 5.82 (d, J=2.21 Hz, 1H) 5.97 (s, 2H)6.10 (dd, J=5.73, 2.43 Hz, 1H) 7.17-7.23 (m, 1H) 7.28-7.37 (m, 2H)7.41-7.46 (m, 1H) 7.80 (d, J=5.73 Hz, 1H). LCMS (ESI): m/z: [M+H] calledfor C₁₃H₁₂N₄O: 241; found 241; RT=2.348 min.

Example 28. Preparation of(S)-2-(((2-aminopyridin-4-yl)thio)methyl)pyrrolidine-1-carbonitrile (28)

To a mixture of compound 28.7 (1 g, 4.97 mmol, 1 eq) and TEA (1.51 g,14.91 mmol, 2.07 mL, 3 eq) in DCM (20 mL) was added methanesulfonylchloride (683.18 mg, 5.96 mmol, 461.61 μL, 1.20 eq) in one portion at 0°C. under N₂. The mixture was stirred at 0° C. for 0.5 hr. The reactionwas monitored by TLC (PE:EtOAc=2:1). The aqueous phase was

extracted with DCM (20 mL×3). The combined organic phase was washed withbrine (5 mL), dried over anhydrous Na₂SO₄, filtered and concentrated invacuum to afford compound 28.6 (1.80 g, crude) was obtained as a blackbrown oil. The crude was used for next step directly. But it didn'twork. The crude was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=10:1 to 2:1) to afford pure compound 28.6 (1.67 g,yield 59.87%).

To a mixture of compound 28.6 (800 mg, 2.86 mmol, 1 eq) in acetone (20mL) was added acetylsulfanylpotassium (327.07 mg, 2.86 mmol, 1 eq) at20° C. under N₂. The mixture was stirred at 50° C. for 15 hours.Filtered and concentrated in vacuum. The aqueous phase was extractedwith EtOAc (20 mL×3). The combined organic phase was washed with brine(5 mL), dried with anhydrous Na₂SO₄, filtered and concentrated invacuum. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=10:1 to 2:1) to afford compound 28.5 (420mg, 1.62 mmol, 56.62% yield) as a colorless oil. ¹H NMR (400 MHz,methanol-d4) ppm 1.49 (s, 9H), 1.76-2.23 (m, 4H), 2.34 (s, 3H),2.96-3.42 (m, 4H), 3.88-3.94 (m, 1H). LCMS (ESI): m/z: [M+H−56] calcdfor C12H22NO3 S: 204; found 204; RT=0.859 min.

To a solution of compound 28.5 (200 mg, 771.13 μmol, 1 eq) in MeOH (5mL) was added NaOH (1 M, 925.36 μL, 1.20 eq) in one portion at 20° C.under N₂. The mixture was stirred at 20° C. for 3 hours. The aqueousphase was extracted with EtOAc (20 mL×3). The combined organic phase waswashed with brine (5 mL), dried with anhydrous Na₂SO₄, filtered andconcentrated in vacuum to give compound 28.4 (100 mg, 460.13 μmol,59.67% yield) as black brown oil. ¹H NMR (400 MHz, DMSO-d6) ppm1.26-1.46 (m, 1H), 1.47 (s, 9H), 1.79-2 (m, 4H), 2.55-2.84 (m, 2H),3.35-3.50 (m, 2H) 3.82-3.88 (m, 1H).

To a mixture of compound 28.4 (100 mg, 460.13 μmol, 1 eq) and compound28.3 (257.61 mg, 690.20 μmol, 1.50 eq), Xanphos (106.50 mg, 184.05 μmol,0.40 eq), DIEA (237.87 mg, 1.84 mmol, 321.45 μL, 4 eq) in 1,4-dioxane (2mL) was added Pd₂(dba)₃ (126.41 mg, 138.04 μmol, 0.30 eq) in one portionat 20° C. under N₂. The mixture was stirred at 80° C. for 15 hours. Thereaction was monitored by LCMS. The aqueous phase was extracted withEtOAc (20 mL×3). The combined organic phase was washed with brine (5mL), dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum togive compound 28.2 (140 mg, crude) was obtained as light yellow oil. ¹HNMR (400 MHz, DMSO-d6) ppm 1.45-1.53 (m, 27H), 1.84-2.04 (m, 4H),2.74-2.79 (m, 1H), 3.37-3.63 (m, 3H), 4.05 (m, 1H), 7.15 (s, 1H), 7.35(m, 1H), 8.30 (m, 1H).

A solution of compound 28.2 (140 mg crude) in HCl/EtOAc (5 mL) wasstirred at 20° C. for 15 hours under N₂. The reaction was monitored byTLC (SiO₂, PE:EtOAc=1:1). After the reaction was completed, the solutionwas concentrated in vacuum to give compound 28.1 (50 mg, 156.89 μmol,79.96% yield, 3HCl) was obtained as a light yellow solid.

To a mixture of compound 28.1 (50 mg, 238.88 μmol, 1 eq) and BrCN (25.30mg, 238.88 μmol, 17.57 μL, 1 eq) in DMF (1 mL) was added DIEA (123.49mg, 955.52 μmol, 166.88 μL, 4 eq) in one portion at 0° C. under N₂. Themixture was stirred at 0° C. for 10 min. The reaction was monitored byLC-MS promptly until the reaction was completed. The crude was purifiedby prep-HPLC (basic condition) to afford product 28 (10 mg, 42.68 μmol,17.87% yield) as light yellow oil. ¹H NMR (400 MHz, DMSO-d6) ppm1.79-1.94 (m, 1H), 1.94-1.98 (m, 2H), 2.11-2.15 (m, 1H), 2.88-2.94 (m,1H), 3.35-3.54 (m, 3H), 3.84-3.85 (m, 1H), 4.48 (s, 2H), 6.40 (s, 1H),6.54 (dd, 7=1.6 Hz, 7=5.6 Hz, 1H), 7.91 (d, 7=5.6 Hz, 1H). LCMS (ESI):m/z: [M+H] calcd for C₁₁H₁₄N₄S: 235; found 235; RT=2.209 min.

Example 29. Preparation of(S)-2-(((2-amino-5-methylpyridin-4-yl)oxy)methyl)pyrrolidine-1-carbonitrile(29)

To a mixture of compound 29.8 (5 g, 24.84 mmol, 1 eq) and4-methylbenzenesulfonyl chloride (5.68 g, 29.81 mmol, 1.20 eq) in DCM(50 mL) was added TEA (3.77 g, 37.26 mmol, 5.16 mL, 1.50 eq) and DMAP(485.62 mg, 3.97 mmol, 0.16 eq) in one portion at 0° C. under N₂. Themixture was then heated to 25° C. and stirred for 10 hours. The reactionmixture was diluted with H₂O 50 mL and extracted with DCM 30 mL (30 mL×

3). The combined organic layers were washed with brine 60 mL (60 mL×1),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive compound 29.7 (8.90 g, crude) as a yellow oil. LCMS (ESI): m/z:[M+H] calcd for C₁₇H₂₅NO₅S: 355; found 300,256; RT=0.890 min.

To a mixture of compound 29.7 (8.84 g, 24.87 mmol, 1 eq) and compound29.6 (3.01 g, 27.36 mmol, 1.10 eq) in DMF (100 mL) was added NaH (895.44mg, 37.31 mmol, 1.50 eq) portionwise at 80° C. under N₂. The mixture wasstirred at 80° C. for 10 hours. The reaction mixture was quenched byaddition H₂O 100 mL, and then extracted with EtOAc 80 mL (80 mL×3). Thecombined organic layers were washed with brine 150 mL (150 mL×1), driedover Na₂SO₄, filtered and concentrated under reduced pressure to givecompound 29.5 (4.50 g, 15.34 mmol, 61.68% yield) as a white solid. ¹HNMR (400 MHz, chloroform-d) δ ppm 1.40 (s, 9H) 1.71-1.95 (m, 4H) 3.28(br s, 2H) 3.63-3.91 (m, 1H) 3.97-4.15 (m, 1H) 4.05 (br dd, J=14.18,7.03 Hz, 1H) 5.85-6.05 (m, 1H) 6.22 (br d, J=4.39 Hz, 1H) 7.81 (br d,J=5.14 Hz, 1H). LCMS (ESI): m/z: [M+H] calcd for C₁₅H₂₃N₃O₂: 293; found294; RT=0.692 min.

To a mixture of compound 29.5 (1 g, 3.41 mmol, 1 eq) in DMF (15 mL) wasadded NBS (606.69 mg, 3.41 mmol, 1 eq) in one portion at 18° C. underN₂. The mixture was stirred at 18° C. for 3 hours. The reaction mixturewas quenched by addition H₂O 15 mL and extracted with EtOAc 10 mL (10mL×3). The combined organic layers were washed with brine 10 mL (10mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give compound 29.4 (1 g, 2.69 mmol, 78.78% yield) as acolorless oil. ¹H NMR (400 MHz, DMSO-dis) ppm 1.38 (br d, 7=4.19 Hz, 9H)1.72-1.83 (m, 1H) 1.93 (br s, 1H) 1.98-2.17 (m, 2H) 3.24-3.31 (m, 2H)3.94-4.10 (m, 4H) 6.02 (s, 2H) 6.10 (s, 1H) 7.72-7.92 (m, 1H). LCMS(ESI): m/z: [M+H] calcd for C₁₅H₂₂N₃BrO₃: 372; found 372,374; RT=0.763min.

To a mixture of compound 29.4 (800 mg, 2.15 mmol, 1 eq) and DMAP (42.01mg, 344 μmol, 0.16 eq), TEA (869.84 mg, 8.60 mmol, 1.19 mL, 4 eq) in THF(15 mL) was added Boc₂O (1.17 g, 5.38 mmol, 1.23 mL, 2.50 eq) in oneportion at 18° C. under N₂. The mixture was stirred at 18° C. for 10hours. The reaction mixture was diluted with H₂O 20 mL and extractedwith EtOAc 30 mL (10 mL×3). The combined organic layers were washed withbrine 20 mL (20 mL×1), dried over Na₂SO₄, filtered and concentratedunder reduced pressure to give a residue. The residue was purified bycolumn chromatography (SiO₂, Petroleum ether/Ethyl acetate=5:1) to givecompound 29.3 (900 mg, 1.57 mmol, 73.02% yield) as a yellow oil. ¹H NMR(400 MHz, chloroform-d) δ ppm 1.40 (s, 27H) 1.81 (br s, 1H) 1.95-2.17(m, 3H) 3.28-3.48 (m, 2H) 4.13 (br s, 3H) 6.70-6.95 (m, 1H) 8.33 (br s,1H). LCMS (ESI): m/z: [M+H] calcd for C₂₅H₃₈N₃BrO₇: 572; found 574,572;RT=18 min.

To a mixture of compound 29.3 (750 mg, 1.59 mmol, 1 eq), Cs2CO3 (2.07 g,6.36 mmol, 4 eq) and methylboronic acid (142.77 mg, 2.38 mmol, 1.50 eq)in DMF (15 mL) was added Pd(dppf)Cl₂ (349.03 mg, 477 μmol, 0.30 eq) inone portion at 80° C. under N₂. The mixture was stirred at 80° C. for 10hours. The reaction mixture was diluted with H₂O 20 mL and extractedwith EA 30 mL (10 mL×3). The combined organic layers were washed withbrine 30 mL (30 mL×1), dried over Na₂SO₄, filtered and concentratedunder reduced pressure to give compound 29.2 (800 mg, crude) as a yellowsolid. LCMS (ESI): m/z: [M+H] calcd for C₂₁H₃₃N₃O₅: 407; found 208;RT=0.150 min.

Compound 29.2 (800 mg, 1.58 mmol, 1 eq) was added into a solution ofHCl/MeOH (15 mL). The mixture was stirred at 18° C. for 10 hours. Thereaction mixture was concentrated under reduced pressure to give aresidue. The residue was purified by prep-HPLC (TFA condition) to givecompound 29.1 (200 mg, 964.92 μmol, 61.07% yield) as a white solid. LCMS(ESI): m/z: [M+H] calcd for C₁₁H₁₇N₃O: 207; found 208; RT=0.173 min.

To a mixture of compound 29.1 (100 mg, 482.46 μmol, 1 eq) and DIPEA(249.41 mg, 1.93 mmol, 337.05 μL, 4 eq) in DMF (3 mL) was added CNBr(56.21 mg, 530.71 μmol, 39.04 μL, 1.10 eq) in one portion at 0° C. underN₂. The mixture was stirred at 0° C. for 10 min. The residue waspurified by prep-HPLC (neutral condition) to give product 29 (10 mg,43.05 μmol, 8.92% yield) as a yellow solid. ¹H NMR (400 MHz,chloroform-d) δ ppm 1.87-2.04 (m, 4H) 2.07 (s, 2H) 2.10-2.21 (m,1H)3.41-3.65 (m, 2H) 3.91-4.12 (m, 2H) 5.95 (s, 1H) 7.72 (s, 1H). LCMS(ESI): m/z: [M+H] calcd for C₁₂H₁₆N₄O: 232; found 233; RT=2.181 min.

Example 30. Preparation of(S)-2-(((2-amino-5-bromopyridin-4-yl)oxy)methyl)pyrrolidine-1-carbonitrile(30)

To a mixture of compound 30.6 (5 g, 24.84 mmol, 1 eq) and4-methylbenzenesulfonyl chloride (5.68 g, 29.81 mmol, 1.20 eq) in DCM(50 mL) was added TEA (3.77 g, 37.26 mmol, 5.16 mL, 1.50 eq) and DMAP(485.62 mg, 3.97 mmol, 0.16 eq) in one portion at 0° C. under N₂. Themixture was then heated to 25° C. and stirred for 10 hours. The reactionmixture was diluted with H₂O 50 mL and extracted with DCM 90 mL (30mL×3). The combined organic layers were washed with brine 60 mL (60mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give compound 30.5 (8.90 g, crude) as a yellow oil. LCMS(ESI): m/z: [M+H] calcd for C₁₇H₂₅NO₅S: 356; found 300; RT=0.890 min.

To a mixture of compound 30.5 (8.84 g, 24.87 mmol, 1 eq) and compound30.4 (3.01 g, 27.36 mmol, 1.10 eq) in DMF (100 mL) was added NaH (895.44mg, 37.31 mmol, 1.50 eq) portionwise at 80° C. under N₂. The mixture wasstirred at 80° C. for 10 hours. The reaction mixture was quenched byaddition H₂O 1000 mL, and then extracted with EtOAc 240 mL (80 mL×3).The combined organic layers were washed with brine 150 mL (150 mL×1),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,DCM/MeOH=10:1) to give compound 30.3 (4.50 g, 15.34 mmol, 61.68% yield)as a white solid. LCMS (ESI): m/z: [M+H] calcd for C₁₅F₂₃N₃O₃: 294;found 294; RT=0.692 min.

To a mixture of compound 30.3 (1 g, 3.41 mmol, 1 eq) in DMF (15 mL) wasadded NBS (606.69 mg, 3.41 mmol, 1 eq) in one portion at 18° C. underN₂. The mixture was stirred at 18° C. for 3 hours. The reaction mixturewas quenched by addition H₂O 15 mL and extracted with EtOAc 30 mL (10mL×3). The combined organic layers were washed with brine 10 mL (10mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Ethyl acetate) to give compound 30.2 (1 g, 2.69mmol, 78.78% yield) as a colorless oil. LCMS (ESI): m/z: [M+H] calcd forC₁₅H₂₂BrN₃O₃: 372; found 372, 374; RT=0.763 min.

Compound 30.2 (100 mg, 268.63 μmol, 1 eq) was added into a solution ofHCl/MeOH (5 mL). The mixture was stirred at 18° C. for 10 hours. Thereaction mixture was concentrated under reduced pressure to givecompound 30.1 (120 mg, 440.95 μmol, 164.15% yield) as a white solid.LCMS (ESI): m/z: [M+H] calcd for C₁₀H₁₄N₃OBr: 272; found 272,274;RT=0.096 min.

To a mixture of compound 30.1 (100 mg, 367.46 μmol, 1 eq) and DIPEA(189.96 mg, 1.47 mmol, 256.70 μL, 4 eq) in DMF (3 mL) was added CNBr(42.81 mg, 404.20 μmol, 29.73 μL, 1.10 eq) in one portion at 0° C. underN₂. The mixture was stirred at 0° C. for 10 min. The residue waspurified by prep-HPLC (TFA condition) to give product 30 (20 mg, 67.31μmol, 18.32% yield) as a yellow solid. LCMS (ESI): m/z: [M+H] calcd forC₁₁H₁₃N₄O: 297; found 297,299; RT=2.364 min.

Example 31. Preparation of(2,S)-2-(2-(2-aminopyridin-4-yl)-1-hydroxyethyl)pyrrolidine-1-carbonitrile(31)

To a solution of compound 31.5 (1 g, 9.25 mmol, 1 eq) in THF (50 mL) wasadded Boc₂O (6.06 g, 27.75 mmol, 6.38 mL, 3 eq), TEA (3.74 g, 37 mmol,5.13 mL, 4 eq) and DMAP (339.02 mg, 2.77 mmol, 0.30 eq) at 25° C. underN₂. The resulting mixture was stirred at 25° C. for 16 hours. Thereaction mixture was added water (30 mL), extracted with EtOAc (50mL×3). The organic phase was separated, washed with saturated NaCl (20mL) and dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=10:1 to 1:1).Compound 31.4 (1.50 g, 4.86 mmol, 52.59% yield) was obtained as a yellowsolid.

To a solution of compound 31.4 (1.50 g, 4.86 mmol, 1 eq) in THF (20 mL)was added t-BuLi (1.3 M, 7.48 mL, 2 eq) drop-wise at −78° C. under N₂.During which the temperature was maintained below −78° C. The reactionmixture was stirred at −78° C. for 1 h. Then compound 31.3 (969.21 mg,4.86 mmol, 1 eq) was added to above mixture. The resulting mixture wasstirred at 25° C. for 16 hours. The reaction mixture was added water (10mL), extracted with EtOAc (20 mL×3). The organic phase was separated,washed with saturated NaCl (10 mL) and dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=10:1 to 1:1). Compound 31.2 (500 mg, 984.99 μmol, 20.27% yield)was obtained as a yellow oil.

To a solution of compound 31.2 (500 mg, 984.99 μmol, 1 eq) in HCl/EtOAc(10 mL) was stirred at 25° C. for 16 hours. The reaction mixture wasconcentrated under reduced pressure to give a residue. Compound 31.1(400 mg, crude) was obtained as a yellow oil.

To a solution of compound 31.1 (400 mg, 1.43 mmol, 1 eq, 2HCl) in DMF (5mL) was added DIEA (553.51 mg, 4.28 mmol, 747.99 μL, 3 eq) and BrCN(151.21 mg, 1.43 mmol, 105.01 μL, 1 eq) at 25° C. under N₂. Theresulting mixture was stirred at 25° C. for 2 hours. The reactionmixture was added water (10 mL), extracted with EtOAc (10 mL×3). Theorganic phase was separated, washed with saturated NaCl (10 mL) anddried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by prep-HPLC (neutralcondition). Product 30 (15 mg, 64.58 μmol, 4.52% yield) was obtained asa white solid. LCMS (ESI): m/z: [M+H] calcd for C₁₂H₁₆N₄O₂:232; found233; RT=2.141, 2.394 min. ¹H NMR (400 MHz, methanol-d4) δ ppm 1.70-2.12(m, 4H) 2.58-2.60 (m, 1H) 2.91-2.98 (m, 1H) 3.46-3.99 (m, 2H) 4.13-4.16(m, 1H) 5.12-5.14 (m, 1H) 6.48-6.58 (m, 2H) 7.78-7.82 (m, 1H)

Example 32. Preparation of(S,E)-2-(2-(2-aminopyridin-4-yl)vinyl)pyrrolidine-1-carbonitrile (32)

To a solution of compound 32.6 (5 g, 14.19 mmol, 1 eq) in THF (50 mL)was added LAH (1.35 g, 35.48 mmol, 2.50 eq) at 0° C. and stirred for 1hour. The mixture was warmed to 25° C. gradually and stirred for 11 hr.The reaction mixture was quenched by addition 8% NaOH solution 2 mL at25° C., and then diluted with H₂O 20 mL and extracted with EtOAc 60 mL(20 mL×3). The combined organic layers were washed with brines 20 mL (20mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=1:1) to givecompound 32.5 (1.50 g, 6.69 mmol, 47.14% yield) as a white solid. LCMS(ESI): m/z: [M+H] calcd for C₁₁H₁₆N₂O₃: 224; found 225; RT=0.404 min.

To a solution of compound 32.5 (1.50 g, 6.69 mmol, 1 eq) in THF (15 mL)was added CBr₄ (3.33 g, 10.03 mmol, 1.50 eq) and PPh₃ (2.63 g, 10.03mmol, 1.50 eq). The mixture was stirred at 25° C. for 2 hours. Thereaction mixture was diluted with H₂O 20 mL and extracted with EtOAc 45mL (15 mL×3). The combined organic layers were washed with brine 20 mL(20 mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=3:1) to givecompound 32.4 (1.10 g, 3.83 mmol, 57.26% yield) as a white solid. 1H NMR(400 MHz, chloroform-d) δ ppm 1.54 (s, 9H) 4.36 (s, 2H) 6.97 (dd,J=5.29, 1.54 Hz, 1H) 7.94-8.08 (m, 1H) 8.29 (d, J=5.29 Hz, 1H) 9.23 (s,1H)

To a solution of compound 32.4 (400 mg, 1.39 mmol, 1 eq) in toluene (7mL) was added PPh₃ (401.04 mg, 1.53 mmol, 1.10 eq) under nitrogen andreflux 120° C. for 3 hours. After cooling to 25° C., the white solid wasfiltered off, washed with EtOAc and dried under vacuum. Then the mixturewas stirred in THF (4 mL) under nitrogen and t-BuOK (171.57 mg, 1.53mmol, 1.10 eq) was added. The mixture was stirred at 70° C. for 1 hr.After cooling to 25° C., compound 32.3 (415.44 mg, 2.09 mmol, 1.50 eq)in THF (1 mL) was added to the reaction flask and reflux for 30 min. Thereaction mixture was diluted with H₂O 15 mL and extracted with EtOAc 45mL (15 mL×3). The combined organic layers were washed with brine 15 mL(15 mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by prep-TLC (SiO₂,Petroleum ether/Ethyl acetate=1:1) to give compound 32.2 (200 mg, 513.49μmol, 36.94% yield) as a brown oil. LCMS (ESI): m/z: [M+H] calcd forC₂₁H₃₁N₃O₄: 389; found 390; RT=1.508 min.

To a solution of compound 32.2 (180 mg, 462.14 μmol, 1 eq) in HCl/EtOAc(5 mL) was stirred at 25° C. for 12 hours. The reaction mixture wasconcentrated under reduced pressure to remove solvent to give compound32.1 (150 mg, crude) as a brown oil. LCMS (ESI): m/z: [M+H] calcd forC₁₁H₁₅N₃:189; found 190; RT=0.382 min.

To a solution of compound 32.1 (150 mg, 792.56 μmol, 1 eq) in DMF (2 mL)was added DIEA (409.72 mg, 3.17 mmol, 553.68 μL, 4 eq) at 0° C. When thepH >9, the mixture was added BrCN (83.95 mg, 792.56 μmol, 58.30 μL, 1eq) and stirred at 0° C. for 10 min. The residue was purified byprep-HPLC (column: YMC-Actus Triart C18 150×30 5u; mobile phase: [water(10 mM NH4HCO3)-ACN]; B %: 10%-30%, 12 min) to give product 32 (5 mg,23.34 μmol, 2.94% yield) as a brown oil. LCMS (ESI): m/z: [M+H] calcdfor C₁₂H₁₄N₄:214; found 215; RT=2.160 min. 1H NMR (400 MHz, methanol-d4)δ ppm 1.63-1.74 (m, 1H) 1.78-2 (m, 3H) 2.03-2.15 (m, 1H) 3.33-3.54 (m,2H) 4.16 (q, J=7.01 Hz, 1H) 6.22-6.30 (m, 1H) 6.42 (s, 1H) 6.45-6.49 (m,1H) 6.62 (d, J=4.52 Hz, 1H) 7.65-7.82 (m, 1H).

Example 33. Preparation of4-(2-aminopyridin-4-yl)piperazine-1-carbonitrile (33)

To a solution of compound 33.5 (1 g, 5.78 mmol, 1 eq) in THF (20 mL) wasadded Boc₂O (3.15 g, 14.45 mmol, 3.32 mL, 2.50 eq), TEA (2.34 g, 23.12mmol, 3.21 mL, 4 eq) and DMAP (211.84 mg, 1.73 mmol, 0.30 eq) at 25° C.under N₂. The result mixture was stirred at 25° C. for 16 hours. Thereaction mixture was added water (20 mL), extracted with EtOAc (20mL×3). The organic phase was separated, washed with saturated NaCl (10mL) and dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=10:1 to 1:1).Compound 33.4 (1 g, 2.68 mmol, 46.35% yield) was obtained as a yellowsolid.

To a solution of compound 33.4 (1 g, 2.68 mmol, 1 eq) in DMF (10 mL) wasadded compound 33.3 (499.01 mg, 2.68 mmol, 1 eq) Pd₂(dba)₃ (245.34 mg,267.92 μmol, 0.10 eq) and CS₂CO₃ (2.62 g, 8.04 mmol, 3 eq) at 25° C.under N₂. The result mixture was stirred at 80° C. for 16 hours. Thereaction mixture was added water (10 mL), extracted with EtOAc (10mL×3). The organic phase was separated, washed with saturated NaCl (10mL) and dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. Compound 33.2 (500 mg, 1.04 mmol, 38.98%yield) was obtained as a white solid. LCMS (ESI): m/z: [M+H] called forC₂₄H₃₈N₄O₆: 479; found 479; RT=0.771 min.

To a solution of compound 33.2 (500 mg, 1.04 mmol, 1 eq) in HCl/EtOAc (5mL) was stirred at 25° C. for 16 hours. The reaction mixture wasconcentrated under reduced pressure to give a residue. Compound 33.1(100 mg, 561.07 μmol, 53.95% yield) was obtained as a white solid.

To a solution of compound 33.1 (100 mg, 398.15 μmol, 1 eq, 2HCl) in DMF(3 mL) was added DIEA (205.83 mg, 1.59 mmol, 278.15 μL, 4 eq) and BrCN(42.17 mg, 398.15 μmol, 29.28 μL, 1 eq). The mixture was stirred at 0°C. for 10 min. The residue was purified by prep-HPLC (neutralcondition). Product 33 (2 mg, 9.84 μmol, 2.47% yield) was obtained as ayellow solid. ¹H NMR (400 MHz, methanol-d₄) δ ppm 3.33-3.38 (m, 4H)3.40-3.46 (m, 4H) δ (s, 1H) 6.22-6.32 (m, 1H) 7.62 (d, J=6.39 Hz, 1H).LCMS (ESI): m/z: [M+H] called for C₁₀H₁₃N₅: 204; found 204; RT=1.133min.

Example 34. Preparation ofN-(1-((2-aminopyridin-4-yl)oxy)-3-methylbutan-2-yl)-N-methylcyanamide(34)

To a solution of compound 34.8 (1 g, 9.69 mmol, 1.06 mL, 1 eq) in THF(15 mL) was added IMIDAZOLE (2.64 g, 38.76 mmol, 4 eq) and TBDPSCl (5.33g, 19.38 mmol, 4.98 mL, 2 eq). The mixture was stirred at 25° C. for 14hours. The reaction mixture was quenched by addition H₂O 50 mL at 25°C., and extracted with EtOAc (50 mL×3). The combined

organic layers were washed with saturated brine (25 mL×1), dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to give a residue. Compound 34.7 (4 g, crude) was obtained as ayellow oil, and the crude product was used into the next step withoutfurther purification.

To a solution of compound 34.7 (4 g, 11.71 mmol, 1 eq) in THF (30 mL)was added TEA (4.74 g, 46.84 mmol, 6.49 mL, 4 eq) and Boc₂O (3.83 g,17.57 mmol, 4.04 mL, 1.50 eq). The mixture was stirred at 25° C. for 14hours. The reaction mixture was quenched by addition H₂O 50 mL at 25°C., and extracted with ethyl acetate (50 mL×3). The combined organiclayers were washed with saturated brine (25 mL×1), dried over anhydroussodium sulfate, filtered and concentrated under reduced pressure to givea residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=5/1). Compound 34.6 (3 g, 6.79 mmol,93.69% yield) was obtained as a colorless oil.

To a solution of compound 34.6 (1 g, 2.26 mmol, 1 eq) in THF (20 mL) wasadded NaH (108.48 mg, 4.52 mmol, 2 eq) the mixture was stirred at 25° C.for 1 hr. Then to the mixture was added Mel (384.94 mg, 2.71 mmol,168.83 μL, 1.20 eq). The mixture was stirred at 0° C. for 12 hours. Thereaction mixture was quenched by addition H₂O 20 mL at 25° C. andextracted with ethyl acetate (20 mL×3). The combined organic layers werewashed with saturated brines (20 mL×1), dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give acrude product. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1). Compound 34.5 (600 mg, 1.32 mmol,58.41% yield) was obtained as a yellow oil.

To a solution of compound 34.5 (600 mg, 1.32 mmol, 1 eq) in THF (15 mL)was added TBAF (345.13 mg, 1.32 mmol, 1 eq) the mixture was stirred at25° C. for 2 hours. The reaction mixture was quenched by addition H₂O 20mL at 25° C. and extracted with ethyl acetate (20 mL×3). The combinedorganic layers were washed with saturated brines (20 mL×1), dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to give a crude product. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1). Compound 34.4(250 mg, 1.15 mmol, 87.16% yield) was obtained as a yellow oil.

To a solution of compound 34.4 (250 mg, 1.15 mmol, 1 eq) in DCM (5 mL)was added TEA (349.11 mg, 3.45 mmol, 478.23 μL, 3 eq) and DMAP (28.10mg, 230 μmol, 0.20 eq) and TosCl (263.10 mg, 1.38 mmol, 1.20 eq). Themixture was stirred at 25° C. for 2 hours. The reaction mixture wasquenched by addition H₂O 20 mL at 25° C. and extracted with ethylacetate (20 mL×3). The combined organic layers were washed withsaturated brines (20 mL×1), dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a crudeproduct. Compound 34.3 (500 mg, crude) was obtained as a yellow oil.

To a solution of compound 34.3 (400 mg, 1.08 mmol, 1 eq) and compound34.3a (118.56 mg, 1.08 mmol, 1 eq) in DMF (10 mL) was added K₂CO₃(595.27 mg, 4.31 mmol, 4 eq). The mixture was stirred at 80° C. for 13hours. The reaction mixture was quenched by addition H₂O 20 mL at 25° C.and extracted with ethyl acetate (20 mL×3). The combined organic layerswere washed with saturated brines (20 mL×1), dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give acrude product. The residue was purified by column chromatography (SiO₂,DCM:MeOH=10:1). Compound 34.2 (200 mg, 646.41 μmol, 60.03% yield) wasobtained as a yellow oil. LCMS (ESI): m/z: [M+H] called for C₁₆H₂₇N₃O₃:310; found 310; RT=0.591 min.

A mixture of compound 34.2 (200 mg, 646.41 μmol, 1 eq) in HCl/EtOAc (15mL) was stirred at 25° C. for 13 hours. The mixture was concentratedunder reduced pressure to give a residue. Compound 34.1 (170 mg, 602.39μmol, 93.19% yield, 2HCl) was obtained as a yellow solid. LCMS (ESI):m/z: [M+H] called for C₁₁H₁₉N₃O: 210; found 210; RT=0.098 min.

To a solution of compound 34.1 (170 mg, 602.39 μmol, 1 eq, 2HCl) in DMF(2 mL) was added DIEA (311.41 mg, 2.41 mmol, 420.83 μL, 4 eq) and BrCN(63.80 mg, 602.39 μmol, 44.31 μL, 1 eq). The mixture was stirred at 0°C. for 10 min. The residue was purified by prep-HPLC (neutralcondition). Product 34 (40 mg, 170.72 μmol, 28.34% yield) was obtainedas a yellow oil. ¹H NMR (400 MHz, methanol-d₄) δ ppm 0.96-1.17 (m, 6H)1.87-2.19 (m, 1H) 2.88-3.07 (m, 4H) 4.02-4.35 (m, 2H) 6.14 (d, J=1.76Hz, 1H) 6.30 (dd, J=6.06, 1.87 Hz, 1H) 7.73 (d, J=5.95 Hz, 1H). LCMS(ESI): m/z: [M+H] called for C₁₂H₁₈N₄O: 235; found 235; RT=2.295 min.

Example 35. Preparation ofN-(1-((2-aminopyridin-4-yl)oxy)butan-2-yl)-N-methylcyanamide (35)

To a solution of compound 35.7 (1 g, 4.76 mmol, 1 eq) and compound 35.6(342.98 mg, 4.76 mmol, 413.23 μL, 1 eq) in DMF (25 mL) was added K₂CO₃(1.97 g, 14.27 mmol, 3 eq). The mixture was stirred at 80° C. for 14hours. The reaction mixture was quenched by addition H₂O 20 mL at 25° C.and extracted with ethyl acetate (20 mL×3). The combined organic layerswere washed with saturated brines (20 mL×1), dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give acrude product. Compound 35.5 (2.5 g, crude) was obtained as a yellowoil. It was used into the next step without further purification.

To a solution of compound 35.5 (2 g, 3.29 mmol, 1 eq) in THF (20 mL) andH₂O (30 mL) was added TEA (1.67 g, 16.46 mmol, 2.28 mL, 5 eq) and Boc₂O(2.16 g, 9.88 mmol, 2.27 mL, 3 eq) and DMAP (120.68 mg, 987.82 μmol,0.30 eq). The mixture was stirred at 25° C. for hours. The reactionmixture was quenched by addition H₂O 20 mL at 25° C. and extracted withethyl acetate (20 mL×3). The combined organic layers were washed withsaturated brines (20 mL×1), dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a crudeproduct. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1:1). Compound 35.4 (230 mg, 814.65 μmol,24.74% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H]called for C₁₄H₂₂N₄O₂: 283; found 283; RT=0.654 min.

To a solution of compound 35.4 (230 mg, 814.65 μmol, 1 eq) in DCM (5 mL)was added Dess-Martin periodinane (691.05 mg, 1.63 mmol, 504.42 μL, 2eq) mixture was stirred at 25° C. for 14 hours. The reaction mixture wasquenched by addition H₂O 20 mL at 25° C. and extracted with ethylacetate (20 mL×3). The combined organic layers were washed withsaturated brines (20 mL×1), dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a crudeproduct. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1). Compound 35.3 (225 mg, 802.65 μmol,98.53% yield) was obtained as a yellow oil. ¹H NMR (400 MHz,chloroform-d) δ ppm 1.13 (t, J=7.28 Hz, 3H) 1.48-1.59 (m, 1H) 1.48-1.59(m, 10H) 2.63 (q, J=7.28 Hz, 2H) 4.65 (s, 2H) 6.53 (dd, J=5.73, 2.43 Hz,1H) 7.58 (d, J=1.98 Hz, 1H) 8.07-8.22 (m, 1H). LCMS (ESI): m/z: [M+H]called for C₁₄H₂₀N₄O₂: 281; found 281; RT=0.615 min.

A mixture of compound 35.3 (100 mg, 356.74 μmol, 1 eq) in MeNH₂ (2 mL)was stirred at 0° C. for 1 hr. To the mixture was added HOAc (32.13 mg,535.10 μmol, 30.60 μL, 1.50 eq) make the mixture to pH=4 and then addedNaBH₃CN (89.67 mg, 1.43 mmol, 4 eq). The mixture was stirred at 25° C.for 12 hours. The reaction mixture was quenched by addition saturatedNaHCO₃ 20 mL at 25° C. make the pH>7 and extracted with ethyl acetate(20 mL×3). The combined organic layers were washed with saturated brines(20 mL×1), dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure to give a crude product. The residuewas purified by column chromatography (SiO₂, DCM:MeOH=10:1). Compound35.2 (80 mg, 270.84 μmol, 75.92% yield) was obtained as a yellow oil. Itwas combined with a second batch for a total of 160 mg. LCMS (ESI): m/z:[M+H] called for C₁₅H₂₅N₃O₃: 296; found 296; RT=0.520 min.

A mixture of compound 35.2 (160 mg, 541.68 μmol, 1 eq) in HCl/EtOAc (5mL) was stirred at 25° C. for 13 hours. The mixture was concentratedunder reduced pressure to give a residue. Compound 35.1 (140 mg, 522.04μmol, 96.38% yield, 2HCl) was obtained as a yellow oil.

To a solution of compound 35.1 (140 mg, 522.04 μmol, 1 eq, 2HCl) in DMF(2 mL) was added DIEA (269.87 mg, 2.09 mmol, 364.69 μL, 4 eq) and BrCN(55.29 mg, 522.04 μmol, 38.40 μL, 1 eq). The mixture was stirred at 0°C. for 10 min. The residue was purified by prep-HPLC (neutralcondition). Product 35 (40 mg, 181.60 μmol, 34.79% yield) was obtainedas a yellow oil. ¹H NMR (400 MHz, methanol-d₄) δ ppm 1.07 (t, J=7.39 Hz,3H) 1.60-1.82 (m, 2H) 2.98 (s, 3H) 3.15-3.30 (m, 1H) 3.97-4.19 (m, 2H)6.13 (d, J=2.21 Hz, 1H) 6.28 (dd, 0.7=6.17, 2.20 Hz, 1H) 7.73 (d,0.7=6.17 Hz, 1H). LCMS (ESI): m/z: [M+H] called for C₁₁H₁₆N₄O: 221;found 221; RT=2.115 min.

Example 36. Preparation ofN-(2-((2-aminopyridin-4-yl)oxy)-1-phenylethyl)-N-methylcyanamide (36)

The solution of 36.7 (5 g, 45.41 mmol, 1 eq) in THF (50 mL) was addedtriethyl amine (18.38 g, 181.64 mmol, 25.18 mL, 4 eq) and Boc₂O (29.73g, 136.23 mmol, 31.29 mL, 3 eq) and DMAP (1.66 g, 13.62 mmol, 0.30 eq).The mixture was stirred at 25° C. for 16 hours. The rethyl acetatectionmixture was quenched by addition H₂O 50 mL at 25° C., and extracted withethyl acetate (50 mL×3). The combined organic layers were washed withsaturated brine (25 mL×1), dried over anhydrous sodium sulfate, filteredand concentrated under reduced pressure to give a residue. The crudeproduct compound 36.6 (4 g, 9.75 mmol) was used into the next stepwithout further purification. LCMS (ESI): m/z: [M+H] calcd forC₂₀H₃₀N₂O₇: 311; found 411; RT=0.941 min.

To a solution of compound 36.6 (3 g, 9.67 mmol, 1 eq) in MeOH (40 mL)and H₂O (4 mL) was added NaOH (966.65 mg, 24.17 mmol, 2.50 eq). Themixture was stirred at 25° C. for 16 hours. The rethyl acetatectionmixture was quenched by addition H₂O 25 mL at 25° C., and extracted withethyl acetate (25 mL×3). The combined organic layers were washed withsaturated brine (25 mL×1), dried over anhydrous sodium sulfate, filteredand concentrated under reduced pressure to give a residue. The residuewas purified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=4/l) to give a compound 36.5 (1.50 g, 7.14 mmol, 73.79% yield)as a white solid. LCMS (ESI): m/z: [M+H] calcd for C₁₀H₁₄O₃N₂: 211;found 211; RT=0.352 min.

To a solution of compound 36.5 (500 mg, 2.38 mmol, 1 eq) in DMF (30 mL)was added K₂CO₃ (986.82 mg, 7.14 mmol, 3 eq) and compound 36.4 (570 mg,2.86 mmol, 1.2 eq). The mixture was stirred at 25° C. for 14 hours. Thereaction mixture was quenched by addition H₂O 20 mL at 25° C., andextracted with ethyl acetate (20 mL×3). The combined organic layers werewashed with saturated brine (20 mL×1), dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give acompound 36.3 (260 mg, 791.81 μmol, crude) as a white solid, and thecrude product was without further purification. LCMS (ESI): m/z: [M+H]calcd for C₁₈H₂₀O₄N₂: 329; found 329; RT=0.701 min.

To a solution of compound 36.3 (200 mg, 609.09 μmol, 1 eq) in NH₂CH₃ (2mL) was added HOAc (54.86 mg, 913.63 μmol, 52.25 μL, 1.50 eq) andNaBH₃CN (153.10 mg, 2.44 mmol, 4 eq). The mixture was stirred at 25° C.for 14 hours. The rethyl acetatection mixture was quenched by additionH₂O 50 mL at 25° C., and extracted with ethyl acetate (50 mL×3). Thecombined organic layers were washed with saturated brine (25 mL×1),dried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=2/l) to give acompound 36.2 (80 mg, 232.95 μmol, 38.25% yield) as a white solid. ¹HNMR (400 MHz, chloroform-d) δ ppm 1.50 (s, 9H) 2.35 (s, 3H) 3.95-4.06(m, 2H) 4.07-4.14 (m, 1H) 6.49 (dd, J=5.70, 2.19 Hz, 1H) 7.28-7.33 (m,1H) 7.34-7.44 (m, 4H) 7.50 (d, J=1.75 Hz, 1H) 8.03 (d, J=5.70 Hz, 1H).LCMS (ESI): m/z: [M+H] calcd for C₁₉H₂₅O₃N₃: 343; found 343; RT=0.587min.

To a solution of compound 36.2 (80 mg, 232.95 μmol, 1 eq) in HCl/EtOAc(15 mL). The mixture was stirred at 25° C. for 14 hours. The rethylacetatection mixture was filtered and concentrated under to give thecompound 36.1 (50 mg, 205.51 μmol, 88.22% yield) as a white solid. LCMS(ESI): m/z: [M+H] calcd for C₁₄H₁₇N₃O: 244; found 244; RT=0.104 min.

To a solution of compound 36.1 (50 mg, 308.26 μmol, 1 eq) in DMF (5 mL)was added BrCN (32.65 mg, 308.26 μmol, 22.67 μL, 1 eq) and DIETHYLACETATE (159.36 mg, 1.23 mmol, 215.35 μL, 4 eq). The mixture was stirredat 0° C. for 0.5 hour. The residue was purified by prep-HPLC (neutralcondition) to give product 36 (25 mg, 93.18 μmol, 45.34% yield) as awhite solid. ¹H NMR (400 MHz, chloroform-d) δ ppm 1.57 (s, 9H) 2.87 (s,3H) 4.14-4.33 (m, 2H) 4.33-4.55 (m, 3H) 5.99 (d, J=1.98 Hz, 2H) 6.27(dd, J=5.95, 2.20 Hz, 1H) 7.36-7.53 (m, 5H) 7.90 (d, J=5.95 Hz, 2H).LCMS (ESI): m/z: [M+H] calcd for C₁₅H₁₆N₄O: 269; found 269; RT=2.835min.

Example 37. Preparation of2-(1-aminoisoquinolin-6-yl)piperidine-1-carbonitrile (37)

A mixture of compound 37.4 (250 mg, 590.60 μmol, 1 eq),tributyl(2-pyridyl)stannane (434.86 mg, 1.18 mmol, 2 eq), Pd(PPh₃)₂Cl₂(41.45 mg, 59.06 μmol, 0.10 eq) in dioxane (2 mL) was degassed andpurged with N₂ for 3 times, and then the mixture was stirred at 80° C.for 10 hour under N₂ atmosphere. TLC (PE:EtOAc=2:1, Rf=0.45) showed thereaction was completed. The reaction mixture was quenched by additionH₂O 50 mL at 25° C., and extracted with ethyl acetate (50 mL×3). Thecombined organic layers were washed with saturated brines (10 mL×1),dried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure to give a crude product. The residue was purified bycolumn chromatography (SiO₂, Petroleum ether/Ethyl acetate=30/l to 1:1)to give compound 37.3 (200 mg, 474.51 μmol, 80.34% yield) as a whitesolid. ¹H NMR (400 MHz, chloroform-d) δ ppm 1.28-1.40 (m, 19H) 7.31-7.38(m, 1H) 7.70-7.98 (m, 3H) 8.08 (d, J=8.80 Hz, 1H) 8.29 (dd, J=8.86, 1.16Hz, 1H) 8.43-8.57 (m, 2H) 8.79 (d, J=4.77 Hz, 1H).

To a solution of compound 37.3 (200 mg, 474.51 μmol, 1 eq) in MeOH (5mL) was added PtO₂ (21.55 mg, 94.90 μmol, 0.20 eq). The mixture wasstirred under H₂ (50 Psi) at 50° C. for 12 hour. The reaction mixturewas concentrated under reduced pressure to give a residue. The residuewas purified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=30/l to 1:1) to give compound 37.2 (150 mg, 350.84 μmol, 73.94%yield) as a white solid. ¹H NMR (400 MHz, methanol-d₄) δ ppm 1.28 (s,14H) 1.61-1.78 (m, 4H) 1.94-2.04 (m, 2H) 2.87 (br t, J=12.13 Hz, 1H)3.23 (br d, J=12.79 Hz, 1H) 3.91 (br d, J=10.14 Hz, 1H) 7.79 (br d,J=8.60 Hz, 1H) 7.87 (d, J=5.73 Hz, 1H) 7.94 (d, J=8.60 Hz, 1H) δ (s, 1H)8.35 (d, J=5.95 Hz, 1H). LCMS (ESI): m/z: [M+H] calcd for C₂₄H₃₃N₃O₄:428; found 428; RT=0.971 min.

To a solution of compound 37.2 (150 mg, 350.84 μmol, 1 eq) was addedHCl/EtOAc (12.81 mg, 350.84 μmol, 2 mL, 1 eq). The mixture was stirredat 25° C. for 3 hours. The reaction mixture was concentrated underreduced pressure to give compound 37.1 (35 mg, 153.98 μmol, 43.89%yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C₁₄H₁₇N₃: 228;found 228; RT=0.131 min.

To a solution of compound 37.1 (35 mg, 153.98 μmol, 1 eq) in DMF (1 mL)was added CNBr (16.31 mg, 153.98 μmol, 11.33 μL, 1 eq) and DIPEA (59.70mg, 461.94 μmol, 80.68 μL, 3 eq). The mixture was stirred at 25° C. for5 min. The residue was purified by prep-HPLC (TFA condition) to giveProduct 37 (10 mg, 39.63 μmol, 25.74% yield) as a white solid. ¹H NMR(400 MHz, DMSO-dis) δ ppm 1.50-1.93 (m, 6H) 3.28 (td, J=11.84, 3.51 Hz,1H) 3.49-3.57 (m, 1H) 4.27 (dd, J=10.52, 3.07 Hz, 1H) 6.50 (br s, 2H)6.93 (d, J=5.70 Hz, 1H) 7.48 (dd, J=8.55, 1.53 Hz, 1H) 7.70 (s, 1H) 7.82(d, J=5.70 Hz, 1H) 8.19 (d, J=8.77 Hz, 1H). LCMS (ESI): m/z: [M+H] calcdfor C₁₅H₁₆N₄: 253; found 253; RT=2.505 min.

Example 38. Preparation of3-((2-aminopyridin-4-yl)thio)pyrrolidine-1-carbonitrile (38)

To a solution of compound 38.2 (90 mg, 181.59 μmol, 1 eq) in HCl/EtOAc(5 mL) at 20° C. under N₂. The mixture was stirred at 20° C. for 4hours. Filtered and concentrated in vacuum to give compound 38.1 (50 mg,164.11 μmol, 90.37% yield, 3HCl) as a light yellow solid.

To a mixture of compound 38.1 (50 mg, 256.04 μmol, 1 eq) andcarbononitridic bromide (27.12 mg, 256.04 μmol, 18.83 μL, 1 eq) in DMF(1 mL) was added DIEA (132.36 mg, 1.02 mmol, 178.87 μL, 4 eq) in oneportion at 0° C. under N₂. The mixture was stirred at 0° C. for 10 min.The residue was purified by prep-HPLC (basic condition) to give Product38 (4.40 mg, 19.97 μmol, 7.80% yield) as a light yellow oil. LCMS (ESI):m/z: [M+H] calcd for C₁₀H₁₂N₄S: 221; found 221; RT=0.943 min. 1H NMR(400 MHz, chloroform-d) δ ppm 1.46 (br s, 5H) 1.90-2 (m, 1H) 2.24-2.36(m, 1H) 3.32 (dd, J=10.23, 4.71 Hz, 1H) 3.46 (td, J=8.63, 5.33 Hz, 1H)3.53-3.62 (m, 1H) 3.77 (dd, J=10.29, 6.53 Hz, 1H) 3.86 (quin, J=5.62 Hz,1H) 4.38 (br s, 2H) 6.28 (d, J=1.13 Hz, 1H) 6.44 (dd, J=5.52, 1.51 Hz,1H) 7.87 (d, J=5.52 Hz, 1H).

Examples 39 and 40. Preparation ofN-(2-((2-aminopyridin-4-yl)oxy)ethyl)-N-isopropylcyanamide (39) andN-(2-((2-aminopyridin-4-yl)oxy)ethyl)-N-cyclohexylcyanamide (40)

To a mixture of compound 39.5 (5 g, 45.41 mmol, 1 eq) and compound 39.4(10.18 g, 45.41 mmol, 1 eq) in DMF (20 mL) was added NaH (2.18 g, 54.49mmol, 60% purity, 1.20 eq) in portions at 0° C. under N₂. The mixturewas stirred at 15° C. for 15 hours. The reaction was monitored by LCMS.After the reaction was completed, water (120 mL) was added dropwise at0° C. The aqueous phase was extracted with EtOAc (30 mL×3). The combinedorganic phase was washed with brine (5 mL), dried with anhydrous Na₂SO₄,filtered and concentrated in vacuum. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=10:1 to 2:1) to givecompound 39.3 (2.80 g, 11.05 mmol, 24.34% yield) was obtained as a whitesolid. ¹H NMR (400 MHz, methanol-d4) ppm 1.45 (s, 9H), 3.52 (d, J=5.2Hz, 2H), 4 (t, J=5.2 Hz, 2H), 4.43 (br, 2H), 5.05 (br, 1H), 5.96 (d,<7=1.6, 1H), 6.25 (dd, <7=1.6 Hz, <7=6.0 Hz, 1H), 7.90 (d, <7=6.0 Hz,1H). LCMS (ESI): m/z: [M+H] calcd for C₁₂H₂₀N₃O₃: 254; found 254;RT=0.585 min.

A solution of compound 39.3 (2.80 g, 11.05 mmol, 1 eq) in HCl/MeOH (40mL) was stirred at 15° C. for 2 hrs under N₂. The mixture wasconcentrated in vacuum to give compound 39.2 (2.40 g, crude, HCl salt)was obtained as a white solid.

To a mixture of compound 39.2 (400 mg, 2.61 mmol, 1 eq) and acetone(379.16 mg, 6.53 mmol, 479.95 μL, 2.50 eq) in CH₃CN (10 mL) was addedNaBH(OAc)₃ (1.38 g, 6.53 mmol, 2.50 eq) at 0° C. under N₂. The mixturewas stirred at 0° C. for 30 mins, then added CH₃COOH (31.36 mg, 522μmol, 29.87 μL, 0.20 eq) at 0° C. and stirred for 1 hr. The reaction wasmonitored by LCMS. Filtered and concentrated in vacuum. Compound 39.1(1.40 g, crude) was obtained as black brown oil. LCMS (ESI): m/z: [M+H]calcd for C₁₀H₁₉N₃O: 196; found 196; RT=0.528 min.

To a mixture of compound 39.1 (400 mg, crude) and BrCN (216.98 mg, 2.05mmol, 150.68 μL, 1 eq) in DMF (1 mL) was added DIEA (1.06 g, 8.19 mmol,1.43 mL, 4 eq) in one portion at 0° C. under N₂. The mixture was stirredat 0° C. for 5 mins. The reaction was monitored by LCMS promptly untilthe reaction was completed. Water (50 mL) was added. The aqueous phasewas extracted with EtOAc (30 mL×3). The combined organic phase waswashed with brine (5 mL), dried with anhydrous Na₂SO₄, filtered andconcentrated in vacuum. The residue was purified by prep-HPLC (basiccondition) to give Product 39 (20 mg, 45.40 μmol, 2.21% yield, 50%purity) was obtained as a colorless oil. ¹H NMR (400 MHz, DMSO-d6) ppm1.30 (d, 7=6.4, 6H), 3.28 (m, 1H), 3.42 (t, J=5.2 Hz, 2H), 4.16 (t,J=5.2 Hz, 2H), 4.68 (br, 2H) 6.01 (d, 7=2.4, 1H), 6.27 (dd, 7=2.4 Hz,7=6.0 Hz, 1H), 7.89 (d, 7=6.0 Hz, 1H). LCMS (ESI): m/z: [M+H] calcd forC11H17N4O: 221; found 221; RT=2.091 min.

To a mixture of compound 39.2 (188 mg, 831.49 μmol, 1 eq, 2HCl) andcyclohexanone 40.2 (244.81 mg, 2.49 mmol, 257.69 μL, 3 eq) in CH3CN (5mL) was added NaBH(OAc)₃ (528.68 mg, 2.49 mmol, 3 eq) in one portion at0° C. under N₂. The mixture was stirred at 0° C. for 30 mins, then addedAcOH (9.99 mg, 166.30 μmol, 9.51 μL, 0.20 eq) at 0° C. and stirred for 1hours. The reaction was monitored by LCMS. Filtered and concentrated invacuum to give compound 40.1 (1.40 g, crude) as black brown oil. LCMS(ESI): m/z: [M+H] calcd for C13H22N3O: 236; found 236; RT=1.159 min.

To a mixture of compound 40.1 (180 mg, 764.92 μmol, 1 eq) and BrCN(81.02 mg, 764.92 μmol, 56.26 μL, 1 eq) in DMF (1 mL) was added DIEA(395.43 mg, 3.06 mmol, 534.37 μL, 4 eq) in one portion at 0° C. underN₂. The mixture was stirred at 0° C. for 12 mins. The reaction wasmonitored by LC-MS promptly until the reaction was completed. Theresidue was purified by prep-HPLC (basic condition) to give product 40(10 mg, 38.41 μmol, 5.02% yield) was obtained as a white solid. ¹H NMR(400 MHz, DMSO-d6) δ ppm 1.18-1.30 (m, 3H), 1.41-1.47 (m, 2H), 1.64-1.67(m, 1H), 1.84-1.87 (m, 2H), 1.99-2.02 (m, 2H), 2.82-2.88 (m, 1H), 3.44(t, 7=4.8 Hz, 2H), 4.14 (t, 7=4.8 Hz, 2H), 4.42 (br. s, 2H), 5.99 (s,1H), 6.26 (d, 7=6.0 Hz, 1H), 7.92 (d, J=6.0 Hz, 1H). LCMS (ESI): m/z:[M+H] calcd for C14H21N4O: 261; found 261; RT=2.546 min.

Examples 41 and 42. Preparation of(S)-2-(((2-amino-5-chloropyridin-4-yl)oxy)methyl)pyrrolidine-1-carbonitrile(41) and(S)-2-(((2-amino-3-chloropyridin-4-yl)oxy)methyl)pyrrolidine-1-carbonitrile(42)

To a mixture of compound 41.6 (5 g, 24.84 mmol, 1 eq) and4-methylbenzenesulfonyl chloride (5.68 g, 29.81 mmol, 1.20 eq) in DCM(50 mL) was added TEA (3.77 g, 37.26 mmol, 5.16 mL, 1.50 eq) and DMAP(485.62 mg, 3.97 mmol, 0.16 eq) in one portion at 0° C. under N₂. Themixture was then heated to 25° C. and stirred for 10 hours. The reactionmixture was diluted with H₂O 50 mL and extracted with DCM 90 mL (30 mL×

3). The combined organic layers were washed with brine 60 mL (60 mL×1),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive compound 41.5 (8.90 g, crude) as a yellow oil. LCMS (ESI): m/z:[M+H] calcd for C₁₇H₂₅NO₅S: 356; found 300; RT=0.890 min.

To a mixture of compound 41.5 (8.84 g, 24.87 mmol, 1 eq) and compound41.4 (3.01 g, 27.36 mmol, 1.10 eq) in DMF (100 mL) was added NaH (895.44mg, 37.31 mmol, 1.50 eq) portionwise at 80° C. under N₂. The mixture wasstirred at 80° C. for 10 hours. The reaction mixture was quenched byaddition H₂O 1000 mL, and then extracted with EtOAc 240 mL (80 mL×3).The combined organic layers were washed with brine 150 mL (150 mL×1),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,DCM/MeOH=10:1) to give compound 41.3 (4.50 g, 15.34 mmol, 61.68% yield)as a white solid. LCMS (ESI): m/z: [M+H] calcd for C₁₅H₂₃N₃O₃: 294;found 294; RT=0.692 min.

To a mixture of compound 41.3 (300 mg, 1.02 mmol, 1 eq) in DMF (5 mL)was added NCS (136.20 mg, 1.02 mmol, 1 eq) in one portion at 20° C.under N₂. The mixture was stirred at 20° C. for 10 hours. The reactionmixture was diluted with H₂O 5 mL and extracted with EtOAc 15 mL (5mL×3). The combined organic layers were washed with brine 10 mL (10mL×1), dried over Na2SO4, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO2, Ethyl acetate) to give compound 41.2 (100 mg,305.05 μmol, 29.91% yield) as a colorless oil. LCMS (ESI): m/z: [M+H]calcd for C₁₅H₂₂ClN₃O₃: 327; found 321; RT=0.912 min. ¹H NMR (400 MHz,methanol-d₄) δ ppm 1.45 (s, 9H) 1.81-1.93 (m, 1H) 2.02-2.22 (m, 4H)3.34-3.47 (m, 2H) 4.13-4.23 (m, 2H) 6.22 (br d, J=17.42 Hz, 1H) 6.49 (brdd, J=12.57, 5.51 Hz, 1H) 7.67-7.79 (m, 1H)

Compound 41.2 (100 mg, 305.05 μmol, 1 eq) was added into a solution ofHCl/MeOH (5 mL). The mixture was stirred at 18° C. for 10 hours. Thereaction mixture was concentrated under reduced pressure to givecompound 41.1 (130 mg, crude) as a yellow solid. LCMS (ESI): m/z: [M+H]calcd for C₁₀H₁₄ClN₃O: 227; found 228; RT=0.096 min.

To a mixture of compound 41.1 (120 mg, 527.03 μmol, 1 eq) and DIPEA(272.45 mg, 2.11 mmol, 368.18 μL, 4 eq) in DMF (3 mL) was added CNBr(61.41 mg, 579.73 μmol, 42.65 μL, 1.10 eq) in one portion at 0° C. underN₂. The mixture was stirred at 0° C. for 10 min. The residue waspurified by prep-HPLC (neutral condition) to give product 41 (12 mg,47.49 μmol, 9.01% yield) and product 42 (12 mg, 47.49 μmol, 9.01% yield)as a yellow oil.

LCMS (ESI): m/z: [M+H] calcd for C₁₁H₁₃N₄OCl: 252; found 253; RT=2.314min. ¹H NMR (400 MHz, chloroform-d) δ ppm 1.90-2.09 (m, 2H) 2.10-2.23(m, 2H) 3.43-3.52 (m, 1H) 3.60 (dt, J=8.75, 6.23 Hz, 1H) 3.98-4.08 (m,2H) 4.09-4.16 (m, 1H) 4.49 (br s, 2H) 6.02 (s, 1H) 7.93 (s, 1H).

LCMS (ESI): m/z: [M+H] calcd for C₁₁H₁₃N₄OCl: 252; found 253; RT=2.226min. ¹H NMR (400 MHz, chloroform-d) δ ppm 1.90-2.09 (m, 2H) 2.10-2.24(m, 2H) 3.42-3.54 (m, 1H) 3.61 (dt, J=9, 6.16 Hz, 1H) 3.99-4.06 (m, 1H)4.07-4.12 (m, 1H) 4.13-4.20 (m, 1H) 4.86 (br s, 2H) 6.31 (d, J=5.77 Hz,1H) 7.90 (d, J=5.77 Hz, 1H).

Examples 43 and 44. Preparation of(R)-2-(1-aminoisoquinolin-6-yl)pyrrolidine-1-carbonitrile (43) and(S)-2-(1-aminoisoquinolin-6-yl)pyrrolidine-1-carbonitrile (44)

To a mixture of compound 43.7 (2 g, 4.72 mmol, 1 eq), compound 43.6(996.01 mg, 4.72 mmol, 1 eq), K₂CO₃ (3.91 g, 28.32 mmol, 6 eq) indioxane (40 mL) and H₂O (10 mL) was added Pd(dppf)Cl₂ (1.04 g, 1.42mmol, 0.30 eq) in one portion at 15° C. under N₂. The mixture wasstirred at 80° C. for 8 hours. The reaction mixture was quenched byaddition H₂O 100 mL at 25° C., and extracted with EtOAc (50 mL×3). Thecombined organic layers were washed with saturated brines (20 mL×2),dried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure to give a crude product. The residue was purified bysilica gel chromatography (Petroleum ether/Ethyl acetate=20/1, 3/1) toafford compound 43.4

(660 mg, 1.30 mmol, 27.54% yield) and compound 43.5 (1 g, 3.23 mmol,68.48% yield) as brown solid. LCMS (ESI): m/z: compound 43.5: [M+H]calcd for C₁₈H₁₉N₃O₂:310; found 310; RT=1.775 min; compound 43.4: [M+H]calcd for C28H35N3O6:510; found 510; RT=3.724 min.

To a mixture of compound 43.5 (1 g, 3.23 mmol, 1 eq) m THF (10 mL (wasadded Boc₂O (3.53 g, 16.16 mmol, 3.71 mL, 5 eq), TEA (1.31 g, 12.93mmol, 1.79 mL, 4 eq), DMAP (394.91 mg, 3.23 mmol, 1 eq) in one portionat 15° C. under N₂. The mixture was stirred at 15° C. for 12 hours. Thereaction mixture was diluted with H₂O 50 mL and extracted with EtOAc 120mL (40 mL×3). The combined organic layers were washed with brine 20 mL(10 mL×2), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by silica gelchromatography (Petroleum ether/Ethyl acetate=20/1, 3/1) to obtaincompound 43.4 (440 mg, 863.44 μmol, 26.71% yield) as brown solid. LCMS(ESI): m/z: [M+H] calcd for C28H35N3O6:510; found 510; RT=1.616 min.

To a mixture of compound 43.4 (1.10 g, 2.16 mmol, 1 eq) in EtOH (30 mL)was added PtO₂ (490.17 mg, 2.16 mmol, 1 eq) in one portion at 15° C.under H2. The mixture was stirred at 15° C., 50 Psi for 12 hours. Thereaction mixture was filtered and concentrated in vacuum. The residuewas purified by silica gel chromatography (Petroleum ether/Ethylacetate=20/l, 3/1) and future purification by SFC to obtain compound43.2 (300 mg, 584.08 μmol, 27.04% yield) and compound 44.2 (300 mg,584.08 μmol, 27.04% yield) as light yellow oil. LCMS (ESI): m/z: [M+H]calcd for C28H39N3O6:514; found 514; RT=1.494 min.

To a mixture of HCl/EtOAc (4 M, 5 mL) in compound 43.2 (300 mg, 584.08μmol, 1 eq) in one portion at 15° C. The mixture was stirred at 15° C.for 12 hour. The reaction solution was concentrated in vacuum to obtaincompound 43.1 (180 mg, crude, 2HCl) was obtained as light yellow solid.LCMS (ESI): m/z: [M+H] calcd for C13H15N3:214; found 214; RT=0.177 min.

To a mixture of compound 43.1 (140 mg, 489.17 μmol, 1 eq, 2HCl) andDIPEA (252.88 mg, 1.96 mmol, 341.73 μL, 4 eq) in DMF (2 mL) was addedBrCN (56.99 mg, 538.09 μmol, 39.58 μL, 1.10 eq) at 0° C. under N2. Themixture was stirred at 0° C. for 10 min. The residue was purified byprep-HPLC (neutral condition) to give product 4.3 (50 mg, 209.83 μmol,42.90% yield) was obtained as light yellow solid. ¹H NMR (400 MHz,chloroform-d) δ ppm 7.97 (d, J=5.86 Hz, 1H) 7.82 (d, J=8.55 Hz, 1H) 7.63(s, 1H) 7.41 (d, J=8.68 Hz, 1H) 7.05 (d, J=5.86 Hz, 1H) 5.11 (br s, 2H)4.82 (t, J=7.03 Hz, 1H) 3.75-3.83 (m, 1H) 3.62-3.69 (m, 1H) 2.43 (dq,0.7=12.65, 6.45 Hz, 1H) 2.07 (dt, 0.7=14.27, 6.98 Hz, 2H) 1.89-1.99 (m,1H). LCMS (ESI): m/z: [M+H] calcd for C14H14N4:239; found 239; RT=2.035min.

To a mixture of compound 44.2 (300 mg, 584.08 μmol, 1 eq) in HCl/EtOAc(4 M, 5 mL) in one portion at 15° C. under N₂. The mixture was stirredat 15° C. for 5 hours. The reaction solution was concentrated in vacuumto obtain compound 44.1 (180 mg, crude, 2HCl) was obtained as lightyellow solid. LCMS (ESI): m/z: [M+H] calcd for C13H15N3:214; found 214;RT=10 min.

To a mixture of compound 44.1 (140 mg, 489.17 μmol, 1 eq, 2HCl), DIPEA(252.88 mg, 1.96 mmol, 341.73 μL, 4 eq) in DMF (2 mL) in one portion at0° C. under N₂. Then added BrCN (56.99 mg, 538.09 μmol, 39.58 μL, 1.10eq) at 0° C. The mixture was stirred at 0° C. for 10 mins. The residuewas purified by prep-HPLC (neutral condition) to give product 44 (40 mg,167.86 μmol, 34.32% yield) was obtained as light yellow solid. ¹H NMR(400 MHz, chloroform-d) δ ppm 7.98 (d, J=5.86 Hz, 1H) 7.82 (d, J=8.55Hz, 1H) 7.63 (s, 1H) 7.41 (d, J=8.43 Hz, 1H) 7.05 (d, 0.7=5.86 Hz, 1H)5.11 (br s, 2H) 4.82 (t, J=7.03 Hz, 1H) 3.75-3.84 (m, 1H) 3.62-3.70 (m,1H) 2.43 (dq, J=12.74, 6.42 Hz, 1H) 2.03-2.13 (m, 2H) 1.89-2 (m, 1H).LCMS (ESI): m/z: [M+H] calcd for C₁₄H₁₄N₄:239; found 239; RT=2.050 min.

Examples 45 and 46. Preparation of2-(1-aminoisoquinolin-6-yl)pyrrolidine-1-carbonitrile (45) andN-(1-(1-aminoisoquinolin-6-yl)ethyl)-N-methylcyanamide (46)

To a solution of compound 45.7 (2.50 g, 10.31 mmol, 1 eq) in NMP (20 mL)was added NH₃.H₂O (22.74 g, 648.87 mmol, 24.99 mL, 62.94 eq). Themixture was stirred at 150° C. for 15 hours. The reaction mixture wasquenched by addition H₂O 50 mL at 25° C. and extracted with ethylacetate (50 mL×3). The combined organic layers were washed withsaturated brines (10 mL×1), dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a crudeproduct. Compound 45.6 (4 g, crude) was obtained as a yellow oil. It wascombined with obtained second batch to obtain 8 g crude which waspurified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=0:1). The purified compound (2 g, 11.20 mmol, 35.72% yield) wasobtained as a yellow oil. ¹H NMR (400 MHz, methanol-d₄) δ ppm 6.91 (d,0.7=6.14 Hz, 1H) 7.58 (dd, J=8.77, 2.19 Hz, 1H) 7.75 (d, 0.7=5.70 Hz,1H) 7.90 (d, J=1.75 Hz, 1H) 8.01 (d, 7=9.21 Hz, 1H). LCMS (ESI): m/z:[M+H] called for C₉H₇N₂Cl: 179; found 179; RT=0.557 min.

To a solution of compound 45.6 (2 g, 8.97 mmol, 1 eq) in THF (40 mL) wasadded TEA (3.63 g, 35.86 mmol, 4.97 mL, 4 eq) and Boc₂O (4.89 g, 22.41mmol, 5.15 mL, 2.50 eq) and DMAP (328.61 mg, 2.69 mmol, 0.30 eq). Themixture was stirred at 25° C. for 15 hours. The reaction mixture wasquenched by addition H₂O 50 mL at 25° C. and extracted with ethylacetate (50 mL×3). The combined organic layers were washed withsaturated brines (20 mL×1), dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a crudeproduct. The residue was purified by re-crystallization from ethylacetate (15 mL) to give the crude product compound 45.5 (2.90 g, 6.85mmol, 76.38% yield) as yellow solid. ¹H NMR (400 MHz, chloroform-d) δppm 1.30-1.34 (m, 1H) 1.32 (s, 17H) 7.57 (d, 7=5.70 Hz, 1H) 7.71 (dd,7=8.77, 1.75 Hz, 1H) 7.83 (d, 7=8.77 Hz, 1H) 8.05 (d, 7=1.75 Hz, 1H)8.45 (d, 7=5.70 Hz, 1H). LCMS (ESI): m/z: [M+H] called for C₁₉H₂₃BrN₂O₄:424; found 424; RT=0.962 min.

To a solution of compound 45.5 (500 mg, 1.18 mmol, 1 eq) and(1-tert-butoxycarbonylpyrrol-2-yl) boronic acid (249 mg, 1.18 mmol, 1eq) in dioxane (20 mL) and H₂O (5 mL) was added Pd(dppf)Cl₂ (259.03 mg,354 μmol, 0.30 eq) and K₂CO3 (489.26 mg, 3.54 mmol, 3 eq). The mixturewas stirred at 80° C. for 14 hours. The reaction mixture was

quenched by addition H₂O 20 mL at 25° C. and extracted with ethylacetate (20 mL×3). The combined organic layers were washed withsaturated brines (20 mL×1), dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a crudeproduct. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1). Compound 45.4 (100 mg, 196.24 μmol,16.63% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H]called for C₁₈H₁₉N₃O₂: 310; found 310; RT=0.734 min.

To a solution of compound 45.4 (400 mg, 1.29 mmol, 1 eq) in THF (10 mL)was added TEA (522.14 mg, 5.16 mmol, 715.26 μL, 4 eq) and Boc₂O (703.86mg, 3.23 mmol, 740.90 μL, 2.50 eq) and DMAP (47.28 mg, 387 μmol, 0.30eq). The mixture was stirred at 25° C. for 14 hours. The reactionmixture was quenched by addition H2O 20 mL at 25° C. and extracted withethyl acetate (20 mL×3). The combined organic layers were washed withsaturated brines (20 mL×1), dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a crudeproduct. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1). Compound 45.3 (160 mg, 313.98 μmol,24.34% yield) was obtained as a yellow oil. ¹H NMR (400 MHz,chloroform-d) δ ppm 1.34 (s, 19H) 6.28-6.33 (m, 1H) 6.37 (dd, J=3.20,1.65 Hz, 1H) 7.45 (dd, J=3.20, 1.65 Hz, 1H) 7.58-7.68 (m, 2H) 7.83 (s,1H) 7.92 (d, J=8.82 Hz, 1H) 8.43 (d, J=5.73 Hz, 1H).

To a solution of compound 45.3 (160 mg, 313.98 μmol, 1 eq) in EtOH (5mL) was added PtO₂ (11 mg) under N₂. The suspension was degassed undervacuum and purged with H₂ several times. The mixture was stirred underH₂ (50 psi) at 25° C. for 12 hours. The mixture was filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=2:1). Compound 45.2 (150 mg, 292.04 μmol, 93.01% yield) wasobtained as a yellow oil. LCMS (ESI): m/z: [M+H] called for C₂₈H₃₉N₃O₆:514; found 514; RT=1.672 min.

A mixture of compound 45.2 (150 mg, 292.04 μmol, 1 eq) in HCl/EtOAc (20mL) was stirred at 25° C. for 14 hours. The mixture was concentratedunder reduced pressure to give a residue. Compound 45.1 (80 mg, 279.52μmol, 95.71% yield, 2HCl) was obtained as a white solid. LCMS (ESI):m/z: [M+H] called for C₁₃H₁₅N₃: 214; found 214; RT=0.175 min.

To a solution of compound 45.1 (80 mg, 279.52 μmol, 1 eq, 2HCl) in DMF(2 mL) was added DIEA (144.50 mg, 1.12 mmol, 195.27 μL, 4 eq) and BrCN(32.57 mg, 307.47 μmol, 22.62 μL, 1.10 eq). The mixture was stirred at0° C. for 10 min. The residue was purified by prep-HPLC (neutralcondition). Product 45 (20 mg, 83.93 μmol, 30.03% yield) was obtained asa yellow solid. ¹H NMR (400 MHz, methanol-d₄) δ ppm 1.84-2.02 (m, 1H)2.03-2.14 (m, 2H) 2.44 (dq, J=12.47, 6.24 Hz, 1H) 3.57-3.70 (m, 1H) 3.80(q, J=7.78 Hz, 1H) 7 (d, J=5.99 Hz, 1 H) 7.49 (d, J=8.68 Hz, 1H) 7.68(s, 1H) 7.75 (d, J=5.99 Hz, 1H) 8.14 (d, J=8.68 Hz, 1H). LCMS (ESI):m/z: [M+H] called for C₁₄H₁₄N₄: 239; found 239; RT=2.277 min.

To a solution of compound 45.5 (500 mg, 1.18 mmol, 1 eq) in dioxane (15mL) was added Pd(PPh₃)₂Cl₂ (82.82 mg, 118 μmol, 0.10 eq) and compound46.6 (852.31 mg, 2.36 mmol, 796.56 μL, 2 eq). The mixture was stirred at80° C. for 14 hours. The reaction mixture was quenched by addition H₂O20 mL at 25° C. and extracted with ethyl acetate (20 mL×3). The combinedorganic layers were washed with saturated brines (20 mL×1), dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to give a crude product. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1). Compound 46.5(450 mg, 1.09 mmol, 92.01% yield) was obtained as a yellow oil. ¹H NMR(400 MHz, chloroform-d) δ ppm 1.21-1.37 (m, 20H) 1.50 (br t, J=7.02 Hz,3H) 3.93-4.09 (m, 2H) 4.43 (br s, 1H) 4.89 (br s, 1H) 7.67 (br d, J=5.26Hz, 1H) 7.88 (q, J=8.77 Hz, 2H) 8.14 (s, 1H) 8.41 (br d, J=5.70 Hz, 1H).LCMS (ESI): m/z: [M+H] called for C₂₃H₃₀N₂O: 415; found 415; RT=0.979min.

A mixture of compound 46.5 (450 mg, 1.09 mmol, 1 eq) in HCl/EtOAc (10mL) was degassed and purged with N₂ for 3 times and then the mixture wasstirred at 25° C. for 15 hours. The mixture was concentrated underreduced pressure to give a residue. Compound 46.4 (230 mg, 1.03 mmol,94.76% yield, HCl) was obtained as a yellow solid. ¹H NMR (400 MHz,methanol-d₄) δ ppm 2.75 (s, 3H) 7.37 (d, J=7.06 Hz, 1H) 7.65 (d, J=7.06Hz, 1H) 8.25 (dd, J=8.82, 1.32 Hz, 1H) 8.53 (br d, J=6.17 Hz, 2H).

To a solution of compound 46.4 (230 mg, 1.24 mmol, 1 eq) in THF (5 mL)was added TEA (501.90 mg, 4.96 mmol, 687.54 μL, 4 eq) and Boc₂O (676.58mg, 3.10 mmol, 712.18 μL, 2.50 eq) and DMAP (45.45 mg, 372 μmol, 0.30eq). The mixture was stirred at 25° C. for 14 hours. The reactionmixture was quenched by addition H₂O 20 mL at 25° C. and extracted withethyl acetate (20 mL×3). The combined organic layers were washed withsaturated brines (10 mL×1), dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a crudeproduct. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1). Compound 46.3 (200 mg, 517.54 μmol,41.74% yield) was obtained as a yellow solid. LCMS (ESI): m/z: [M+H]called for C₂₁H₂₆N₂O₅: 387; found 287; RT=0.626 min.

A mixture of compound 46.3 (100 mg, 258.77 μmol, 1 eq) in MeNH₂ (2 mL)was stirred at 0° C. for 1 hour. To the mixture was added NaBH₃CN (65.04mg, 1.04 mmol, 4 eq) and then added HOAc (23.31 mg, 388.16 μmol, 22.20μL, 1.50 eq) make the mixture to pH=4. The mixture was stirred at 25° C.for 12 hours. The reaction mixture was quenched by addition saturatedNaHCO₃ 20 mL at 25° C. make the pH>7 and extracted with ethyl acetate(20 mL×3). The combined organic layers were washed with saturated brines(20 mL×1), dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure to give a crude product. The residuewas purified by column chromatography (SiO₂, DCM:MeOH=10:1). Compound46.2 (50 mg, 124.53 μmol, 48.12% yield) was obtained as a yellow oil.LCMS (ESI): m/z: [M+H] called for C₂₂H₃₁N₃O₄: 302; found 302; RT=0.565min.

A mixture of compound 46.2 (50 mg, 165.90 μmol, 1 eq) in HCl/EtOAc (20mL) was stirred at 25° C. for 13 hours. The mixture was concentratedunder reduced pressure to give a residue. Compound 46.1 (45 mg, 164.12μmol, 98.93% yield, 2HCl) was obtained as a yellow oil. LCMS (ESI): m/z:[M+H] called for C₁₂H₁₅N₃: 202; found 202; RT=0.116 min.

To a solution of compound 46.1 (45 mg, 164.12 μmol, 1 eq, 2HCl) in DMF(2 mL) was added DIEA (84.84 mg, 656.48 μmol, 114.65 μL, 4 eq) and BrCN(17.38 mg, 164.12 μmol, 12.07 μL, 1 eq). The mixture was stirred at 0°C. for 10 mins. The residue was purified by prep-HPLC (neutralcondition). Product 46 (5 mg, 22.10 μmol, 13.46% yield) was obtained asa yellow solid. ¹H NMR (400 MHz, methanol-d₄) δ ppm 1.67 (d, J=7.06 Hz,3H) 2.79 (s, 3H) 4.33 (q, J=6.98 Hz, 1H) 7 (d, J=5.95 Hz, 1H) 7.53 (d,J=8.60 Hz, 1H) 7.68 (s, 1H) 7.76 (d, J=5.95 Hz, 1H) 8.16 (d, J=8.60 Hz,1H). LCMS (ESI): m/z: [M+H] called for C₁₃H₁₄N₄: 227; found 227;RT=2.247 min.

Example 47. Preparation of3-(((2-aminopyridin-4-yl)oxy)methyl)pyrrolidine-1-carbonitrile (47)

To a solution of compound 47.4 (800 mg, 7.27 mmol, 1 eq) in DCM (40 mL)was added compound 47.3 (1.71 g, 7.27 mmol, 1 eq), PPh₃ (2.86 g, 10.90mmol, 1.50 eq) and DIAD (2.20 g, 10.90 mmol, 2.12 mL, 1.50 eq) in turnat 0° C. under N₂. The resulting mixture was stirred at 25° C. for 16hours. The reaction mixture was added water 20 mL, extracted with EtOAc(20 mL×3). The combined organic layers were washed with brine (20 mL×1),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1:1). Compound 47.2 (700 mg, 2.14 mmol,29.45% yield) was obtained as a white solid. ¹H NMR (400 MHz,chloroform-d) ppm 2.06-2.10 (m, 1H) 2.66-2.72 (m, 1H) 3.27-3.29 (m, 1H)3.46-3.68 (m, 3H) 3.87-3.94 (m, 5H) 4.43 (s, 2H) 5.15 (s, 2H) 5.95 (d,J=2.0 Hz, 1H) 6.24 (dd, J=6.0 Hz, 2.0 Hz, 1H) 7.30-7.37 (m, 5H) 7.89 (d,J=6.0 Hz, 1H).

H₂ was bubbled into a solution of compound 47.2 (400 mg, 1.22 mmol, 1eq) and H₂ in MeOH (40 mL) at 25° C. under 50 psi for 10 hours. Thereaction mixture was filtered, the filter was concentrated under reducedpressure to remove solvent. Compound 47.1 (200 mg, 1.03 mmol, 84.83%yield) was obtained as a yellow oil. ¹H NMR (400 MHz, methanol-d4) ppm1.58-1.63 (m, 1H) 2.02-2.04 (m, 1H) 2.60-2.79 (m, 2H) 2.94-3.14 (m, 3H)3.91-3.98 (m, 2H) 6.09 (d, J=2.0 Hz, 1H) 6.23 (dd, J=6.0 Hz, 2.0 Hz, 1H)7.69 (d, J=6.4 Hz, 1H).

To a solution of compound 47.1 (100 mg, 517.46 μmol, 1 eq) in THF (5 mL)was added DIEA (133.75 mg, 1.03 mmol, 180.75 μL, 2 eq) and BrCN (54.81mg, 517.46 μmol, 38.06 μL, 1 eq) in turn at 0° C. under N₂. Theresulting mixture was stirred at 0° C. for 1 hour. The reaction mixturewas added water 10 mL, extracted with EtOAc (10 mL×3). The combinedorganic layers were washed with brine (20 Ml×1), dried over Na₂SO₄,filtered and concentrated under reduced pressure to give a residue. Theresidue was purified by p-TLC (SiO₂, Ethyl acetate=0:1). Product 47 (14mg, 64.15 μmol, 12.40% yield) was obtained as a white solid. LCMS (ESI):m/z: [M+H] calcd for C₁₁H₁₄N₄O:218; found 219; RT=2.522 min. ¹H NMR (400MHz, chloroform-d) ppm 1.85-1.88 (m, 1H) 2.12-2.17 (m, 1H) 2.73-2.75 (m,1H) 3.32-3.35 (m, 1H) 3.50-3.63 (m, 3H) 3.92-3.98 (m, 2H) δ (s, 2H) 6.04(s, 1H) 6.27 (dd, J=6.0 Hz, 2.0 Hz, 1H) 7.85 (d, J=6.0 Hz, 1H).

Example 48. Preparation ofN-((7-aminofuro[2,3-c]pyridin-2-yl)methyl)-N-methylcyanamide (48)

To a solution of compound 48.9 (10 g, 77.20 mmol, 1 eq) in DCM (100 mL)was added chloro(methoxy)methane (7.46 g, 92.64 mmol, 7.04 mL, 1.20 eq)and DMAP (1.51 g, 12.35 mmol, 0.16 eq) and DIPEA (19.95 g, 154.40 mmol,26.96 mL, 2 eq) at 0° C. The mixture was stirred at 10° C. for 12 hours.The reaction mixture was diluted with H₂O 30 mL and extracted with DCM90 mL (30 mL×3). The combined organic layers were washed with brine 30mL (30 mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=20/l to 3:1) to givecompound 48.8 (13.20 g, 76.04 mmol, 98.50% yield) as a colorless solid.LCMS (ESI): m/z: [M+H] calcd for C₇H₈ClNO₂: 173; found 174; RT=0.937min. ¹H NMR (400 MHz, chloroform-d) δ ppm 3.51 (s, 3H) 5.23-5.30 (m, 2H)7.18 (dd, J=8.11, 4.60 Hz, 1H) 7.47 (dd, J=8.11, 1.53 Hz, 1H) 8.04 (dd,J=4.82, 1.32 Hz, 1H).

To a solution of compound 48.8 (4.40 g, 25.35 mmol, 1 eq) in THF (130mL) was added dropwise t-BuLi (1.3 M, 48.74 mL, 2.50 eq) at −78° C. Themixture was stirred at −78° C. for 1 hour. Then I₂ (11.58 g, 45.62 mmol,9.19 mL, 1.80 eq) was added above mixture at −78° C. The mixture wasstirred at 10° C. for 12 hours. The reaction mixture was quenched byaddition H₂O 10 mL at 0° C., and then diluted with H₂O 50 mL andextracted with EtOAc 150 mL (50 mL×3). The combined organic layers werewashed with brine 50 mL (50 mL×1), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=20/l to 3:1) to give compound 48.7 (12.80 g, 42.74 mmol, 56.21%yield) as a yellow solid. ¹H NMR (400 MHz, chloroform-d) δ ppm 3.68-3.82(m, 3H) 5.18-5.27 (m, 2H) 7.70 (d, J=5.01 Hz, 1H) 7.80 (d, J=5.01 Hz,1H).

To a solution of compound 48.7 (3.90 g, 13.02 mmol, 1 eq) in THF (100mL) was added ethyl compound 48.6 (1.53 g, 15.63 mmol, 1.53 mL, 1.20 eq)and TMEDA (18.01 g, 154.96 mmol, 23.39 mL, 11.90 eq). The mixture wasdegassed with N₂ for 10 mins. Then the mixture was added CuI (496.01 mg,2.60 mmol, 0.20 eq) and Pd(PPh₃)₄ (3.01 g, 2.60 mmol, 0.20 eq). Then themixture was stirred at 60° C. for 15 hours. The reaction mixture wasdiluted with H₂O 40 mL and extracted with EtOAc 180 mL (60 mL×3). Thecombined organic layers were washed with brine 60 mL (60 mL×1), driedover Na₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=20/l to 5:1) to give compound 48.5 (940mg, 4.17 mmol, 16.01% yield) as a white solid.

To a solution of compound 48.5 (710 mg, 3.15 mmol, 1 eq) in THF (2 mL)was added LiBH₄ (102.80 mg, 4.72 mmol, 1.50 eq) and stirred at 0° C. for12 hours. The reaction mixture was diluted with H₂O 15 mL and extractedwith EtOAc 45 mL (15 mL×3). The combined organic layers were washed withbrine 15 mL (15 mL×1), dried over Na₂SO₄, filtered and concentratedunder reduced pressure to give a residue. The residue was purified bycolumn chromatography (SiO₂, Petroleum ether/Ethyl acetate=2/1 to 1:1)to give compound 48.4 (300 mg, 1.63 mmol, 51.88% yield) as a colorlesssolid. LCMS (ESI): m/z: [M+H] calcd for C8H6ClNO2:183; found 184;RT=0.593 min.

To a solution of compound 48.4 (150 mg, 817.04 μmol, 1 eq) in DCM (2 mL)was added TosCl (186.92 mg, 980.45 μmol, 1.20 eq), DMAP (19.96 mg,163.41 μmol, 0.20 eq) and Et₃N (248.03 mg, 2.45 mmol, 339.76 μL, 3 eq)at 0° C. The mixture was stirred at 10° C. for 2 hours. The reactionmixture was diluted with H₂O 10 mL and extracted with DCM 30 mL (10mL×3). The combined organic layers were washed with brine 10 mL (10mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give compound 48.3 (130 mg, crude) as a yellow solid. LCMS(ESI): m/z: [M+H] calcd for C15H12ClNO4S:337; found 338; RT=0.842 min.

To a solution of compound 48.3 (130 mg, 384.87 μmol, 1 eq) in THF (2 mL)was added MeNH₂ (11.95 mg, 384.87 μmol, 2 mL, 1 eq). The mixture wasstirred at 80° C. for 12 hours. The reaction mixture was diluted withH₂O 10 mL and extracted with EtOAc 30 mL (10 mL×3). The combined organiclayers were washed with brine 10 mL (10 mL×1), dried over Na₂SO₄,filtered and concentrated under reduced pressure to give compound 48.2(50 mg, crude) as a yellow oil. LCMS (ESI): m/z: [M+H] calcd forC9H9ClN2O:196; found 197; RT=0.152 min.

To a solution of compound 48.2 (50 mg, 254.28 μmol, 1 eq) in i-PrOH (7mL) was added NH₂NH₂.H₂O (2.40 g, 48.01 mmol, 2.33 mL, 188.80 eq). Themixture was stirred at 90° C. for 72 hours. The reaction mixture wasconcentrated under reduced pressure to remove solvent to give compound48.1 (60 mg, crude) as a yellow oil.

To a solution of compound 48.1 (60 mg, 338.60 μmol, 1 eq) in DMF (2 mL)was added BrCN (35.86 mg, 338.60 μmol, 24.91 μL, 1 eq) and DIPEA (131.28mg, 1.02 mmol, 176.93 μL, 3 eq). The mixture was stirred at 0° C. for0.2 hour. The residue was purified by prep-HPLC to give product 48 (2mg, 9.89 μmol, 2.92% yield) as a colorless oil. LCMS (ESI): m/z: [M+H]calcd for C10H10N4O:202; found 203; RT=2.484 min. ¹H NMR (400 MHz,methanol-d4) δ ppm 2.95 (s, 3H) 4.43 (s, 2H) 6.75-6.98 (m, 2H) 7.69 (d,J=5.62 Hz, 1H).

Example 49. Preparation of(2R,4R)-2-(1-aminoisoquinolin-6-yl)-4-hydroxypyrrolidine-1-carbonitrile(49)

To a solution of compound 49.7 (1.5 g, 6.19 mmol, 1 eq) in NMP (15 mL)was added NH₃.H₂O (54.58 g, 389.32 mmol, 15 mL, 25% purity, 62.94 eq).The mixture was stirred at 150° C. for 15 hours. The reaction mixturewas quenched by addition of H₂O (500 mL) at 25° C. and extracted withEtOAc (500 mL×3). The combined organic layers were washed with saturatedbrine 50 mL (50 mL), dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure to give a crude product. The residuewas purified by column chromatography (SiO₂, DCM:MeOH=10:1) to givecompound 49.6 (1.6 g, 7.17 mmol, 38.65% yield) as yellow oil.

To a solution of compound 49.6 (1.6 g, 7.17 mmol, 1 eq) in THF (20 mL)was added Boc₂O (6.26 g, 28.69 mmol, 6.59 mL, 4 eq) dropwise at 18° C.under N₂. Then to the mixture was added TEA (3.63 g, 35.86 mmol, 4.99mL, 5 eq) and DMAP (140.20 mg, 1.15 mmol, 0.16 eq). This mixture wasstirred at 18° C. for 10 hours. The reaction mixture was concentratedunder reduced pressure to give a residue. The residue was purified bycolumn chromatography (SiO₂, Petroleum ether/Ethyl acetate=1:1) to givecompound 49.5 (1.1 g, 2.60 mmol, 36% yield) as a white solid. LCMS (ESI)m/z: [M+H] calcd for C₇H₈N₂O₂: 423; found 424; RT=1.501 min.

To a mixture of compound 49.5 (1.1 g, 2.60 mmol, 1 eq), compound 49.4(901.38 mg, 3.90 mmol, 1.5 eq), Ir[dF(CF₃)ppy]₂(dtbbpy)PF₆ (2.81 mg, 26μmol, 0.01 eq), NiCl₂.Pglyme (56.31 mg, 259.86 μmol, 0.1 eq), dtbbpy(104.08 mg, 389.79 μmol, 0.15 eq) in DMF (40 mL) was added CS₂CO₃ (1.27g, 3.90 mmol, 1.5 eq) in one portion in the glovebox. Then the mixturewas irradiated with two 34 W fluorescent lamps for 72 hours at 25° C.The residue was purified by prep-HPLC (TFA conditions) to give compound49.2 (60 mg, 113.3 μmol, 4.4% yield) and compound 49.3 (100 mg, 188.8μmol, 7.3% yield) as yellow solid.

Compound 49.3 (100 mg, 188.81 μmol, 1 eq) was dissolved into HCl/EtOAc(10 mL), the mixture was stirred at 15° C. for 10 hours. The reactionmixture was concentrated under reduced pressure to give compound 49.1(60 mg, crude) as a white solid.

To a mixture of compound 49.1 (60 mg, 261.7 μmol, 1 eq) and cyanogenbromide (27.7 mg, 261.7 μmol, 19.25 μL, 1 eq) in DMF (1 mL) was addedDIPEA (101.5 mg, 785.5 μmol, 136.75 μL, 3 eq) in one portion at 0° C.under N₂. The mixture was stirred at 0° C. for 10 mins. The residue waspurified by prep-HPLC (neutral conditions) to give 49, (4 mg, 15.7 μmol,6% yield) as a white solid. LCMS (ESI) m/z: [M+H] calcd. for C₁₄H₁₄N₄O:254; found 255; RT=1.77 min. ¹H NMR (400 MHz, METHANOL-d4) δ: 1.88-1.99(m, 1H), 2.47-2.58 (m, 1H), 3.57 (d, J=10.51 Hz, 1H), 4 (dd, J=10.51,3.91 Hz, 1H), 4.52 (br s, 1H), 5.59 (dd, J=10.09, 6.42 Hz, 1H), 7.13 (d,J=6.24 Hz, 1H), 7.55-7.61 (m, 1H), 7.82 (d, J=6.36 Hz, 2H) and 8.12 (d,J=8.44 Hz, 1H) ppm.

Examples 50a and 50b. Preparation of(2$4S)-2-(1-aminoisoquinolin-6-yl)-4-hydroxypyrrolidine-1-carbonitrile(50a) and(2R,4S)-2-(1-aminoisoquinolin-6-yl)-4-hydroxypyrrolidine-1-carbonitrile(50b)

To a solution of compound 50.5 (1.5 g, 6.19 mmol, 1 eq) in NMP (15 mL)was added NH₃.H₂O (54.58 g, 389.32 mmol, 15 mL, 63 eq). The mixture wasstirred at 150° C. for 15 hours. The reaction mixture was quenched byaddition H₂O (500 mL) at 25° C. and extracted with EtOAc (500 mL×3). Thecombined organic layers were washed with saturated brine (50 mL), driedover anhydrous sodium sulfate, filtered and concentrated under

reduced pressure to give a crude product. The residue was purified bycolumn chromatography (SiO₂, DCM:MeOH=10:1) to give compound 50.4 (1.6g, 7.17 mmol, 39% yield) as yellow oil.

To a solution of compound 50.4 (1.6 g, 7.17 mmol, 1 eq) in THF (20 mL)was added Boc₂O (6.26 g, 28.69 mmol, 6.59 mL, 4 eq) dropwise at 18° C.under N₂. Then the mixture was added TEA (3.63 g, 35.86 mmol, 4.99 mL, 5eq) and DMAP (140.2 mg, 1.15 mmol, 0.16 eq), and stirred at 18° C. for10 hours. The reaction mixture was concentrated under reduced pressureto give a residue. The residue was purified by column chromatography(SiO₂, Petroleum ether/Ethyl acetate=1:1) to give compound 50.3 (1.5 g)as a white solid. LCMS (ESI) m/z: [M+H] calcd for C₇H₈N₂O₂: 423; found424; RT=1.501 min.

To a mixture of compound 50.3 (1.5 g, 3.54 mmol, 1 eq),(2R,4S)-1-tert-butoxycarbonyl-4-hydroxy-pyrrolidine-2-carboxylic acid(1.23 g, 5.32 mmol, 1.5 eq), Ir[dF(CF₃)ppy]₂(dtbbpy)PF₆ (3.83 mg, 35.44μmol, 0.01 eq), NiCl₂.Pglyme (76.79 mg, 354.36 μmol, 0.1 eq), dtbbpy(141.92 mg, 531.54 μmol, 0.15 eq) in DMF (40 mL) was added CS₂CO₃ (1.73g, 5.32 mmol, 1.5 eq) in one portion in the glovebox. Then the mixturewas irradiated with two 34 W fluorescent lamps for 72 hours at 25° C.The residue was purified by prep-HPLC (TFA condition) to give compound50.2a (100 mg, 188.81 μmol, 5% yield) and compound 50.2b (100 mg, 188.81μmol, 5% yield) as yellow solid.

To a solution of compound 50.2a (100 mg, 188.81 μmol, 1 eq) in DCM (5mL) was added TFA (1 mL) in one portion at 18° C. under N₂. The mixturewas stirred at 18° C. for 10 hours. The reaction mixture wasconcentrated under reduced pressure to give compound 50.1a (110 mg) as awhite solid. ¹H NMR (400 MHz, METHANOL-d4) δ: 2.41-2.62 (m, 2H),3.38-3.46 (m, 1H), 3.67-3.77 (m, 2H), 4.76 (br t, J=3.75 Hz, 1H), 5.19(br dd, J=11.91, 6.39 Hz, 1H), 7.25 (d, J=7.06 Hz, 1H), 7.63 (d, J=7.06Hz, 1H), 7.88 (dd, J=8.71, 1.65 Hz, 1H), 8.02-8.10 (m, 1H) and 8.54 (d,J=8.82 Hz, 1H) ppm.

To a mixture of compound 50.1a (110 mg, 192.53 μmol, 1 eq) and cyanogenbromide (20.39 mg, 192.53 μmol, 14.16 μL, 1 eq) in DMF (1 mL) was addedDIPEA (74.65 mg, 577.58 μmol, 100.6 μL, 3 eq) in one portion at 0° C.under N₂. The mixture was stirred at 0° C. for 10 mins. The residue waspurified by prep-HPLC (neutral condition) to give 50a (5 mg, 19.66 μmol,10% yield) as a white solid. LCMS (ESI) m/z: [M+H] calcd for C₁₄H₁₅N₄O:255; found 255; RT=1.787 min. ¹H NMR (400 MHz, METHANOL-d4) δ: 1.98-2.09(m, 1H), 2.38 (dd, J=13.59, 6.14 Hz, 1H), 3.52 (d, J=10.52 Hz, 1H), 3.98(dd, J=10.52, 3.51 Hz, 1H), 4.52 (br s, 1H), 5.06 (dd, J=10.52, 6.14 Hz,1H), 7.01 (d, J=6.14 Hz, 1H), 7.54 (dd, J=8.77, 1.75 Hz, 1H), 7.74 (dd,J=3.73, 1.97 Hz, 2H) and 8.17 (d, J=8.77 Hz, 1H) ppm.

To a solution of compound 50.2b (100 mg, 188.81 μmol, 1 eq) in DCM (5mL) was added TFA (1 mL) in one portion at 18° C. under N₂. The mixturewas stirred at 18° C. for 10 hours. The reaction mixture wasconcentrated under reduced pressure to give compound 50.1b (110 mg,crude) as a white solid. ¹H NMR (400 MHz, METHANOL-d4) δ: 2.46-2.55 (m,1H), 2.58-2.68 (m, 1H), 3.40-3.49 (m, 1H), 3.68 (dd, J=12.24, 3.86 Hz,1H), 4.79 (br t, J=3.64 Hz, 1H), 5.66 (br dd, J=11.69, 6.39 Hz, 1H),7.46-7.54 (m, 1H), 7.69-7.76 (m, 1H), 7.84-7.93 (m, 1H), 8.19-8.27 (m,1H) and 8.52-8.60 (m, 1H) ppm.

To a mixture of compound 50.1b (110 mg, 192.53 μmol, 1 eq, 3 TFA) andcyanogen bromide (20.39 mg, 192.53 μmol, 14.16 μL, 1 eq) in DMF (1 mL)was added DIPEA (74.65 mg, 577.58 μmol, 100.6 μL, 3 eq) in one portionat 18° C. under N₂. The mixture was stirred at 18° C. for 10 mins. Theresidue was purified by semi-preparative scale HPLC (TFA condition) togive 50b (5 mg, 19.7 μmol, 10% yield) as a white solid. LCMS (ESI) m/z:[M+H] calcd for C₁₄H₁₅N₄O: 255; found 255; RT=1.779 min. ¹H NMR (400MHz, METHANOL-d4) δ: 1.94 (ddd, J=13.48, 9.98, 4.17 Hz, 1H), 2.53 (dd,J=13.59, 6.58 Hz, 1H), 3.53-3.60 (m, 1H), 4 (dd, J=10.74, 3.73 Hz, 1H),4.52 (br s, 1H), 5.60 (dd, J=10.09, 6.58 Hz, 1H), 7.14 (d, J=6.58 Hz,1H), 7.58 (t, J=7.89 Hz, 1H), 7.80-7.87 (m, 2H) and 8.13 (d, J=8.33 Hz,1H) ppm.

Examples 51a and 51b. Preparation of(S)-2-(1-(methylamino)isoquinolin-6-yl)pyrrolidine-1-carbonitrile (51a)and (R)-2-(1-(methylamino)isoquinolin-6-yl)pyrrolidine-1-carbonitrile(51b)

To a solution of compound 51.8 (2.5 g, 10.31 mmol, 1 eq) in NMP (30 mL)was added NH₃.H₂O (30 mL, 33% aqueous solution) in one portion at 150°C. under N₂. The mixture was stirred at 150° C. for 10 hours. Thereaction mixture was diluted with H₂O (500 mL) and extracted with EtOAc(500 mL×3). The combined organic layers were washed with brine (500 mL),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1:2) to give compound 51.7 (35 g, 95%yield) as a white solid. LCMS (ESI) m/z: [M+H] calcd for C₉H₈BrN₂: 223;found 223; RT=0.761 min.

To a solution of compound 51.7 (7 g, 31.4 mmol, 1 eq) in DMF (70 mL) wasadded NaH (1.26 g, 31.4 mmol, 60% suspension in mineral oil, 1 eq)portion-wise at 0° C. under N₂. The mixture was stirred at 0° C. for 30mins, then the mixture was added CH₃I (6.68 g, 47.1 mmol, 2.93 mL, 1.5eq) in one portion, and heated to 25° C. and stirred for 1.5 hours. Thereaction mixture was quenched by addition of H₂O (70 mL) and then wasextracted with EtOAc (70 mL×3). The combined organic layers were washedwith H₂O (70 mL×3), and then with brine (70 mL), dried over Na₂SO₄,filtered and concentrated. This residue was purified by medium pressureliquid chromatography (MPLC) (SiO₂, petroleum ether/ethyl acetate=10/1to 0:1) to give compound 51.5 (2.8 g, 11.81 mmol, 38% yield) as anorange solid and to give compound 51.6 (1.09 g, 4.34 mmol, 14% yield) asyellow oil. ¹H NMR (400 MHz, chloroform-d) δ: 8.03 (d, J=5.70 Hz, 1H),7.82 (d, J=1.75 Hz, 1H), 7.56-7.61 (m, 1H), 7.47-7.53 (m, 1H), 6.82 (d,J=5.70 Hz, 1H), 5.33 (br s, 1H), 3.58 (s, 1H) and 3.15 (d, J=4.82 Hz,3H) ppm.

A mixture of compound 51.5 (2.7 g, 11.4 mmol, 1 eq),N-(t-butoxycarbonyl)pyrrole)-2-boronic acid (2.64 g, 12.53 mmol, 1.1eq), Pd(dppf)Cl₂.CH₂Cl₂ (2.79 g, 3.42 mmol, 0.3 eq), K₂CO₃ (9.44 g,68.33 mmol, 6 eq) in dioxane (28 mL) and H₂O (7 mL) was degassed andpurged with N₂ 3 times; then the mixture was stirred at 80° C. for 5hour under N₂ atmosphere. The reaction mixture was quenched by additionH₂O (15 mL) and then extracted with EtOAc (15 mL×3). The combinedorganic layers were washed with

saturated brine (40 mL), dried over Na₂SO₄, filtered and concentratedunder reduced pressure to give a residue. The residue was purified byMPLC (SiO₂, petroleum ether/ethyl acetate=10/l to 1:1) to give compound51.4 (3.6 g, 11.13 mmol, 98% yield) as a yellow solid. LCMS (ESI) m/z:[M+H] calcd for C₁₉H₂₂N₃O₂: 324; found 324; RT=0.974 min. ¹H NMR (400MHz, chloroform-d) δ: 8.03 (d, J=5.95 Hz, 1H), 7.62-7.72 (m, 2H),7.37-7.49 (m, 1H), 7.37-7.49 (m, 1H), 7.37-7.49 (m, 1H), 7.26 (s, 2H),6.89-6.95 (m, 1H), 6.92 (d, J=5.95 Hz, 1H), 6.23-6.34 (m, 2H), 5.27 (brs, 1H), 3.18 (d, J=4.85 Hz, 3H) and 1.34 (s, 9H) ppm.

To a solution of compound 51.4 (3.60 g, 11.13 mmol, 1 eq) in MeOH (400mL) was added PtO₂ (252.79 mg, 1.11 mmol, 0.1 eq) under N₂. Thesuspension was degassed under vacuum and purged with hydrogen gasseveral times, then was stirred under H₂ (50 psi) at 50° C. for 10hours. The reaction mixture was filtered; the filtrate was concentratedunder reduced pressure to give a residue. The residue was purified byMPLC (SiO₂, petroleum ether/ethyl acetate=10/1 to 1:1) to give compound51.3 (1.8 g, 4.64 mmol, 38% yield, 84.4% purity) as a white solid. LCMS(ESI) m/z: [M+H] calcd for C₁₉H₂₆N₃O₂: 328; found 328; RT=0.899 min. ¹HNMR (400 MHz, chloroform-d) δ: 8.02 (br d, J=5.38 Hz, 1H), 7.69 (br d,J=8.56 Hz, 1H), 7.43 (d, J=1.34 Hz, 1H), 7.29 (br d, J=1.22 Hz, 1H),6.88 (d, J=5.87 Hz, 1H), 5.31 (br s, 1H), 4.79-5.12 (m, 1H), 3.69 (br s,2H), 3.17 (br d, J=4.28 Hz, 3H), 2.39 (br s, 1H), 1.80-2 (m, 4H) and1.09-1.60 (m, 11H) ppm.

500 mg of compound 51.3 was purified by SFC (column: Chiral Pak IC-H250×30 5p; mobile phase: [0.1% NH₃ in H₂O/ETOH]; B %: 40%-40%, 8 min) togive compound 51.2a (188 mg) and compound 51.2b (229 mg) as white solid.

To compound 51.2a (188 mg, 574.18 μmol, 1 eq) was added HCl/EtOAc (4 M,18.6 mL) the reaction mixture was stirred at 25° C. for 12 h. Thereaction mixture was concentrated under reduced pressure to givecompound 51.1a (180 mg) as a hydrogen chloride salt. ¹H NMR (400 MHz,METHANOL-d₄) δ: 8.51 (d, J=8.68 Hz, 1H), 8.06 (s, 1H), 7.89 (br d,J=8.44 Hz, 1H), 7.63 (d, J=6.85 Hz, 1H), 7.26 (d, J=6.85 Hz, 1H),3.45-3.66 (m, 2H), 3.24 (s, 3H), 2.63 (br d, J=8.07 Hz, 1H) and2.16-2.40 (m, 3H) ppm.

To a solution of 51.1a (30 mg, 113.74 μmol, 1 eq) in DMF (1 mL) wasadded cyanogen bromide (12.05 mg, 113.74 μmol, 8.37 μL, 1 eq) and DIPEA(29.4 mg, 227.48 μmol, 39.62 μL, 2 eq). The mixture was stirred at 0° C.for 1 h. The reaction mixture was filtered, and the organic layers hasthe desired compound. The residue was purified by semi-preparative scaleHPLC (column: Agela Durashell C18 150×25 5μ; mobile phase: [water (10 mMNH₄HCO₃)-ACN]; B %: 20%-60%, 10 min) to give 51a (5 mg, 19.4 μmol, 17%yield, 97.9% purity) as a yellow solid. LCMS (ESI) m/z: [M+H] calcd forC₁₅H₁₇N₄:253; found 253; RT=2.357 min. ¹H NMR (400 MHz, METHANOL-d₄) δ:8.10 (d, J=8.60 Hz, 1H), 7.82 (d, J=6.17 Hz, 1H), 7.67 (d, J=1.54 Hz,1H), 7.48 (dd, J=8.60, 1.76 Hz, 1H), 6.92 (d, J=5.73 Hz, 1H), 4.83-4.88(m, 1H), 3.81 (dt, J=8.93, 7.22 Hz, 1H), 3.65 (ddd, J=8.93, 7.39, 5.73Hz, 1H), 3.06 (s, 2H), 2.45 (dq, J=12.43, 6.29 Hz, 1H), 2.04-2.15 (m,2H) and 1.89-1.99 (m, 1H) ppm.

To compound 51.2b (229 mg, 699.41 μmol, 1 eq) was added HCl/EtOAc (4 M,22.7 mL) the reaction mixture was stirred at 25° C. for 12 h. Thereaction mixture was concentrated under reduced pressure to givecompound 51.1b (289 mg) as a hydrogen chloride salt. ¹H NMR (400 MHz,METHANOL-d₄) δ: 8.50 (d, 7=8.68 Hz, 1H), 8.05 (d, 7=1.47 Hz, 1H), 7.89(dd, 7=8.68, 1.83 Hz, 1H), 7.63 (d, 7=6.97 Hz, 1H), 7.26 (d, 7=6.97 Hz,1H), 3.44-3.62 (m, 2H), 3.24 (s, 3H), 2.58-2.67 (m, 1H) and 2.21-2.37(m, 3H) ppm.

To a solution of compound 51.1b (30 mg, 113.74 μmol, 1 eq) in DMF (1 mL)was added cyanogen bromide (12.05 mg, 113.74 μmol, 8.37 μL, 1 eq) andDIPEA (29.40 mg, 227.48 μmol, 39.62 μL, 2 eq). The mixture was stirredat 0° C. for 1 h. The reaction mixture was filtered, and the organiclayers were concentrated. The residue was purified by semi-preparativescale HPLC (column: Agela Durashell C18 150×25 5μ; mobile phase: [water(10 mM NH₄HCO₃)-ACN]; B %: 20%-60%, 10 min) to give 51b (5 mg, 19.7μmol, 17% yield, 99.4% purity) as a white solid. LCMS (ESI) m/z: [M+H]calcd for C₁₅H₁₇N₄: 253; found 253; RT=2.357 min. ¹H NMR (400 MHz,METHANOL-d₄) δ: 8.10 (d, 7=8.77 Hz, 1H), 7.82 (d, 7=6.14 Hz, 1H), 7.66(d, 7=1.75 Hz, 1H), 7.48 (dd, 7=8.77, 1.75 Hz, 1H), 6.92 (d, 7=6.14 Hz,1H), 4.82-4.87 (m, 1H), 3.75-3.86 (m, 1H), 3.60-3.71 (m, 1H), 3.05 (s,3H), 2.38-2.50 (m, 1H), 2.04-2.17 (m, 2H) and 1.89-1.99 (m, 1H) ppm.

Examples 52a and 52b. Preparation of(S)-2-(1-(dimethylamino)isoquinolin-6-yl)pyrrolidine-1-carbonitrile(52a) and(R)-2-(1-(dimethylamino)isoquinolin-6-yl)pyrrolidine-1-carbonitrile(52b)

To a solution of compound 51.3 (800 mg, 2.44 mmol, 1 eq) in DMF (8 mL)was added NaH (134.97 mg, 3.37 mmol, 60% purity, 1.38 eq) portion-wiseat 0° C. under N₂. The mixture was stirred at 0° C. for 30 mins, then tothe mixture was added CH₃I (478.59 mg, 3.37 mmol, 209.91 μL, 1.38 eq) inone portion, and the mixture was heated to 25° C. and stirred for 15.5hours. The reaction mixture was quenched by addition H₂O (10 mL) andextracted with EtOAc (10 mL×3). The combined organic layers were washedwith H₂O (10 mL×3), and the combined organic layers were washed withsaturated brine (10 mL), dried over Na₂SO₄, filtered and concentratedunder reduced pressure to give a residue. The residue was purified byMPLC (SiO₂, petroleum ether/ethyl acetate=10/1 to 1:1) to give compound52.3 (480 mg, 1.33 mmol, 54% yield, 94.4% purity) as yellow oil. LCMS(ESI) m/z: [M+H] calcd for C₂₀H₂₈N₃O₂: 342; found 342; RT=1.170 min. ¹HNMR (400 MHz, chloroform-d) δ: 8.06 (br d, J=8.60 Hz, 3H), 7.46 (s, 1H),7.30 (br d, J=8.82 Hz, 2H), 7.10 (d, J=5.73 Hz, 2H), 4.91 (br s, 1H),3.68 (br s, 3H), 3.09 (br s, 9H), 2.38 (br s, 2H), 1.91 (br t, J=6.06Hz, 5H), 1.46 (br s, 5H) and 1.13 (br s, 9H) ppm.

Compound 52.3 was purified by SFC (column: Chiral Pak AY-H 250×30 5μ;mobile phase, A: 0.1% NH₃H₂O in IP A; B: 30%-30%, 5 min) to givecompound 52.2a (180 mg) and compound 52.2b (200 mg) as white solid.

To compound 52.2a (180 mg, 527.17 μmol, 1 eq) was added HCl/EtOAc (4 M,6 mL).

The reaction mixture was stirred at 25° C. for 12 h. The reactionmixture was concentrated under reduced pressure to give to give compound52.1a (189 mg) as a hydrogen chloride salt. ¹H NMR (400 MHz,METHANOL-d₄) δ: 8.62 (d, J=8.80 Hz, 1H), 8.14 (d, J=1.59 Hz, 1H), 7.91(dd, J=8.86, 1.77 Hz, 1H), 7.71 (d, J=6.85 Hz, 1H), 7.43 (d, J=6.85 Hz,1H), 3.60 (s, 6H), 3.33 (dt, J=3.24, 1.68 Hz, 2H), 2.60-2.73 (m, 1H),2.24-2.45 (m, 3H) and 2.04 (s, 1H) ppm.

To a solution of compound 52.1a (30 mg, 108 μmol, 1 eq, HCl) in DMF (1mL) was added cyanogen bromide (11.44 mg, 108 μmol, 7.94 μL, 1 eq) andDIPEA (27.91 mg, 215.99 μmol, 37.62 μL, 2 eq). The mixture was stirredat 0° C. for 1 h. The reaction mixture was filtered, and the organiclayers were concentrated. The residue was purified by semi-preparativescale HPLC (column: Agela Durashell C18 150×25 5p; mobile phase A:[water (10 mM MH₄HCO₃)-ACN]; B: 25%-65%, 10 min) to give 52a (7 mg,25.42 μmol, 24% yield, 96.7% purity) as a white solid. LCMS (ESI) m/z:[M+H] calcd for C₁₆H₁₉N₄:267; found 267; RT=2.628 min. ¹H NMR (400 MHz,METHANOL-d₄) δ: 8.21 (d, J=8.82 Hz, 1H), 7.97 (d, J=5.95 Hz, 1H), 7.76(d, 0.7=1.54 Hz, 1H), 7.53 (dd, J=8.82, 1.76 Hz, 1H), 7.25 (d, J=5.73Hz, 1H), 4.87-4.90 (m, 1H), 3.81 (dt, 0.7=8.82, 7.28 Hz, 1H), 3.65 (ddd,J=9.04, 7.28, 5.73 Hz, 1H), 3.10 (s, 5H), 3.04-3.14 (m, 1H), 2.39-2.50(m, 1H), 2.03-2.14 (m, 2H) and 1.94 (dq, 0.7=12.49, 7.60 Hz, 1H) ppm.

To compound 52.2b (200 mg, 585.7 μmol, 1 eq) was added HCl/EtOAc (4 M, 6mL). The reaction mixture was stirred at 25° C. for 12 h. The reactionmixture was concentrated under reduced pressure to give compound 52.1b(203 mg) as a hydrogen chloride salt. ¹H NMR (400 MHz, METHANOL-d₄) δ:8.57-8.62 (m, 1H), 8.10-8.13 (m, 1H), 7.85-7.91 (m, 1H), 7.65-7.70 (m,1H), 7.37-7.43 (m, 1H), 3.57 (s, 6H), 3.34 (s, 1H), 2.56-2.74 (m, 1H)and 2.18-2.42 (m, 3H) ppm.

To a solution of compound 52.1b (30 mg, 108 μmol, 1 eq) in DMF (1 mL)was added cyanogen bromide (11.44 mg, 108 μmol, 7.94 μL, 1 eq) and DIPEA(27.91 mg, 216 μmol, 37.62 μL, 2 eq). The mixture was stirred at 0° C.for 1 h. The reaction mixture was filtered, and the organic layers wereconcentrated to give the residue. The residue was purified bysemi-preparative scale HPLC (column: Agela Durashell C18 150×25, 5μ;mobile phase: [water (10 mM NH₄HCO₃)-ACN]; B %: 25%-65%, 10 min) to give52b (7 mg, 26 μmol, 24% yield, 99.8% purity) as a white solid. LCMS(ESI) m/z: [M+H] calcd for C₁₆H₁₉N₄: 267; found 267; RT=2.621 min. ¹HNMR (400 MHz, METHANOL-d₄) δ: 8.22 (d, J=8.77 Hz, 1H), 7.98 (d, J=5.70Hz, 1H), 7.76 (s, 1H), 7.54 (dd, J=8.55, 1.97 Hz, 1H), 7.26 (d, J=6.14Hz, 1H), 4.90 (br s, 1H), 3.78-3.86 (m, 1H), 3.61-3.70 (m, 1H), 3.10 (s,6H), 2.42-2.49 (m, 1H), 2.05-2.15 (m, 1H) and 1.92-2 (m, 1H) ppm.

Example 53. Preparation ofN-(1-(6-aminopyridin-3-yl)-3-oxo-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)cyclopentanecarboxamide(53)

To a solution of compound 53.9 (10 g, 83.95 mmol, 1 eq) in THF (150 mL)was added LAH (6.37 g, 167.90 mmol, 2 eq) at 0° C. The mixture wasstirred at 0° C. for 1.5 hours. The reaction mixture was quenched byaddition saturated sodium sulfate at 0° C. and added 100 ml H₂O thenextracted with ethyl acetate (100 mL×3). The combined organic layerswere washed with saturated brines (30 mL×1), dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give theproduct compound 53.8 (7.50 g, 61.42 mmol, 73.16% yield) as a yellowoil. LCMS (ESI): m/z: [M+H] called for C₆H₆N₂O: 123; found 123; RT=0.099min.

To a solution of compound 53.8 (7.50 g, 61.42 mmol, 1 eq) in THF (100mL) was added TEA (24.86 g, 245.68 mmol, 34.05 mL, 4 eq), Boc₂O (40.21g, 184.26 mmol, 42.33 mL, 3 eq) and DMAP (1.50 g, 12.28 mmol, 0.20 eq).The mixture was stirred at 25° C. for 1.5 hours. The reaction mixturewas quenched by addition H₂O 100 mL at 25° C. and extracted with ethylacetate (100 mL×3). The combined organic layers were washed withsaturated brines (50 mL×1), dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a crudeproduct. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=5:1). Compound 53.7 (1.2 g, 3.72 mmol,6.06% yield) was obtained as a white solid. ¹H NMR (400 MHz,chloroform-d) δ ppm 1.42-1.61 (m, 21H) 7.66 (d, 7=8.38 Hz, 1H) 8.19 (dd,7=8.60, 2.21 Hz, 1H) 8.87 (d, 7=2.21 Hz, 1H) 9.98-10.13 (m, 1H).

To a solution of compound 53.7 (1.03 g, 3.10 mmol, 1 eq) in DCM (20 mL)was added DBU (708.40 mg, 4.65 mmol, 701.39 μL, 1.50 eq). The mixturewas stirred at 25° C. for 0.5 hr. Then to the mixture was added compound53.6 (1 g, 3.10 mmol, 1 eq), the mixture was stirred at 25° C. for 0.5hr. The mixture was concentrated under reduced pressure to give aresidue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1:1). Compound 53.5 (1 g, 1.90 mmol,61.15% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H]called for C₂₇H₃₃N₃O₈: 528; found 528; RT=0.917 min.

To a solution of compound 53.5 (1 g, 1.90 mmol, 1 eq) in MeOH (10 mL)was added Pd—C (10%, 0.3 g) under N₂. The suspension was degassed undervacuum and purged with H₂ several times. The mixture was stirred underH₂ (50 psi) at 25° C. for 15 hours. The reaction mixture was filteredand the filter was concentrated. Compound 53.4 (1 g, crude) was obtainedas a yellow oil. ¹H NMR (400 MHz, chloroform-d) δ ppm 1.35-1.54 (m, 23H)2.83-3.13 (m, 2H) 3.70-3.77 (m, 4H) 7.19 (d, J=8.16 Hz, 1H) 7.61 (dd,J=8.16, 2.43 Hz, 1H) 8.33 (d, J=2.21 Hz, 1H). LCMS (ESI): m/z: [M+H]called for C₁₉H₂₉N₃O₆: 396; found 396; RT=0.701 min.

To a solution of compound 53.4 (600 mg, 1.52 mmol, 1 eq) in DCM (10 mL)was added TEA (307.06 mg, 3.03 mmol, 420.63 μL, 2 eq) andcyclopentanecarbonyl chloride (241.41 mg, 1.82 mmol, 221.48 μL, 1.20eq). The mixture was stirred at 25° C. for 5 mins. The mixture wasconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=1:1). Compound 53.3 (650 mg, 1.32 mmol, 86.99% yield) wasobtained as a yellow oil. LCMS (ESI): m/z: [M+H] called for C₂₅H₃₇N₃O₇:492; found 492; RT=0.866 min.

To a solution of DIPA (349.94 mg, 3.46 mmol, 486.03 μL, 5 eq) in THF (5mL) was added n-BuLi (2.5 M, 1.38 mL, 5 eq). The mixture was stirred at0° C. for 0.5 hr under N₂. Then the mixture was added to the solution ofcompound 53.3 (340 mg, 691.0 μmol, 1 eq) and chloroiodomethane (609.96mg, 3.46 mmol, 251.01 μL, 5 eq) in THF (5 mL) was stirred at −78° C. for0.5 hr. The reaction mixture was quenched by addition saturated NH₄Cl(20 ml) at 25° C. and extracted with ethyl acetate (15 mL×3). Thecombined organic layers were washed with saturated Na₂SO₃ (10 mL) andsaturated NaHCO₃ (10 mL) and saturated brines (10 mL×1), dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to give a crude product. Compound 53.2 (500 mg, crude) wasobtained as a yellow oil. LCMS (ESI): m/z: [M+H] called forC₂₀H₂₈N₃O₄Cl: 410; found 410; RT=0.805 min.

To a solution of compound 53.2 (500 mg, 1.22 mmol, 1 eq) in DMF (5 mL)was added DIEA (472.93 mg, 3.66 mmol, 639.10 μL, 3 eq) and 2, 3, 5,6-tetrafluorophenol (303.85 mg, 1.83 mmol, 1.50 eq). The mixture wasstirred at 25° C. for 15 hours. The reaction mixture was quenched byaddition saturated NaHCO₃ 20 mL at 25° C. and extracted with ethylacetate (20 mL×3). The combined organic layers were washed withsaturated brines (10 mL×1), dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a crudeproduct. The residue was purified by prep-HPLC (TFA condition). Compound53.1 (50 mg, 74.14 umol, 6.08% yield, 80% purity) was obtained as ayellow solid. LCMS (ESI): m/z: [M+H] called for C₂₆H₂₉N₃O₅F₄: 540; found540; RT=1.310 min.

The solution of compound 53.1 (50 mg, 92.67 umol, 1 eq) in HCl/EtOAc (2mL) was stirred at 25° C. for 15 hours. The mixture was concentratedunder reduced pressure to give a residue. The residue was purified byprep-HPLC (TFA condition). Product 53 (5 mg, 11.38 μmol, 12.28% yield)was obtained as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.31-1.77(m, 8H) 2.34 (dt, 7=3.69, 1.79 Hz, 1H) 2.67-2.71 (m, 1H) 3.01 (dd,7=14.11, 4.41 Hz, 1H) 4.60 (ddd, 7=10.20, 8.10, 4.63 Hz, 1H) 5.22 (d,7=2.21 Hz, 2H) 6.89 (d, 7=9.04 Hz, 1H) 7.61 (tt, 7=10.91, 7.28 Hz, 1H)7.71 (d, 7=1.54 Hz, 1H) 7.79 (br d, 7=9.04 Hz, 2H) 8.27 (d, 7=8.16 Hz,1H). LCMS (ESI): m/z: [M+H] called for C₂₁H₂₁N₃O₃F₄: 440; found 440;RT=2.662 min.

Example 54. Preparation ofN-(1-(2-aminopyridin-4-yl)-3-oxo-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)cyclopentanecarboxamide(54)

To a solution of compound 54.12 (58 g, 419.92 mmol, 1 eq) in MeOH (600mL) was added SOCl₂ (99.92 g, 839.84 mmol, 60.93 mL, 2 eq) dropwise at0° C. under N₂. The mixture was stirred at 0° C. for 30 mins, then washeated to 18° C. and stirred at 18° C. for 14.5 hours. The reactionmixture was concentrated under reduced pressure to give compound 54.11(65 g, crude) as a white solid. LCMS (ESI): m/z: [M+H] calcd forC₁H₈N₂O₂: 152; found 153; RT=0.100 min.

To a mixture of methyl compound 54.11 (65 g, 427.21 mmol, 1 eq) and DMAP(2.61 g, 21.36 mmol, 0.05 eq) in t-BuOH (500 mL) and ACETONE (150 mL)was added Boc₂O (279.72 g, 1.28 mol, 294.44 mL, 3 eq) dropwise at 18° C.under N₂. The mixture was stirred at 18° C. for 15 hours. The solutionwas diluted with pentane (200 ml), cooled in the refrigerator for 3hours and filtered to obtain compound 54.10 (110 g, 312.16 mmol, 73.07%yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C₁₇H₂₄N₂O₆:352; found 353; RT=0.877 min. ¹H NMR (400 MHz, chloroform-d) δ ppm1.39-1.50 (m, 19H) 3.97 (s, 3H) 7.77 (dd, J=5.02, 1.38 Hz, 1H) 7.82 (s,1H) 8.62 (d, J=5.02 Hz, 1H).

To a solution of compound 54.10 (60 g, 170.27 mmol, 1 eq) in THF (1 L)was added LiAlH₄ (12.92 g, 340.54 mmol, 2 eq) portionwise at 0° C. underN₂. The mixture was stirred at 0° C. for 1 hours, then heated to 18° C.and stirred at 18° C. for 14 hours. The reaction mixture was quenched byaddition 8% NaOH (15 ml), filtered and then diluted with H₂O 1000 mL andextracted with EtOAc 1500 mL (500 mL×3). The combined organic layerswere washed with brine 1000 mL (1000 mL×1), dried over Na₂SO₄, filteredand concentrated under reduced pressure to give a residue. The residuewas purified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=2:1) to give compound 54.9 (15 g, 66.89 mmol, 39.28% yield) as awhite solid. LCMS (ESI): m/z: [M+H] calcd for C₁₁H₁₆N₂O₃: 293; found294; RT=0.313 min.

To a solution of compound 54.9 (8 g, 35.67 mmol, 1 eq) in DCM (60 mL)was added Dess-Martin periodinane (18.16 g, 42.81 mmol, 13.25 mL, 1.20eq) portionwise at 18° C. under N₂. The mixture was stirred at 18° C.for 2 hours. The reaction mixture was diluted with H₂O 60 mL andextracted with DCM 150 mL (50 mL×3). The combined organic layers werewashed with brine 100 mL (100 mL×1), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=5:1) to give compound 54.8 (5.10 g, 22.95 mmol, 64.33% yield) asa white solid. ¹H NMR (400 MHz, chloroform-d) δ ppm 1.59 (s, 9H) 7.41(dd, J=5.08, 1.32 Hz, 1H) 8.48 (s, 1H) 8.52 (d, J=5.15 Hz, 1H) 8.83 (brs, 1H).

To a solution of compound 54.7 (1.49 g, 4.50 mmol, 1 eq) in DCM (15 mL)was added DBU (1.03 g, 6.75 mmol, 1.02 mL, 1.50 eq) in one portion at18° C. under N₂. The mixture was stirred at 18° C. for 0.5 hour, then tothe mixture was added compound 54.8 (1 g, 4.50 mmol, 1 eq) in oneportion at 18° C., then was stirred at 18° C. for 0.5 hour. The reactionmixture was concentrated under reduced pressure to give a residue. Theresidue was purified by column chromatography (SiO2, Petroleumether/Ethyl acetate=1:1) to give compound 54.6 (1.90 g, 4.44 mmol,98.78% yield) as a white solid.

To a solution compound 54.6 (1 g, 2.34 mmol, 1 eq) in MeOH (50 mL) andTHF (50 mL) was added Pd—C (10%, 100 mg) under N₂. The suspension wasdegassed under vacuum and purged with H₂ several times. The mixture wasstirred under H₂ (50 psi) at 20° C. for 15 hours. The reaction mixturewas filtered and concentrated under reduced pressure to give compound54.5 (700 mg, crude) as a white solid. LCMS (ESI): m/z: [M+H] calcd forC₁₄H₁₉N₃O₄: 293; found 294; RT=0.638 min.

To a solution of compound 54.5 (600 mg, 2.05 mmol, 1 eq) in MeOH (50 mL)and THF (50 mL) was added Pd—C (10%, 100 mg) under N₂. The suspensionwas degassed under vacuum and purged with H₂ several times. The mixturewas stirred under H₂ (50 psi) at 20° C. for hours. The reaction mixturewas filtered and concentrated under reduced pressure to give compound54.4 (600 mg, crude) as a white solid. LCMS (ESI): m/z: [M+H] calcd forC₁₄H₂₁N₃O₄: 295; found 296; RT=0.239 min.

To a mixture of cyclopentanecarboxylic acid (193.24 mg, 1.69 mmol,184.04 μL, 1 eq) and EDCI (357.01 mg, 1.86 mmol, 1.10 eq) in DMF (10 mL)was added HOBt (251.64 mg, 1.86 mmol, 1.10 eq) in one portion at 0° C.under N₂. The mixture was stirred at 0° C. for 1 hour, then the mixturewas added dropwise a solution of compound 54.4 (500 mg, 1.69 mmol, 1 eq)in DMF (5 mL), then the mixture was added dropwise DIPEA (656.42 mg,5.08 mmol, 887.05 μL, 3 eq) and stirred at 0° C. for 1 hour. Thereaction mixture was diluted with H₂O 10 mL and extracted with EtOAc 15mL (5 mL×3). The combined organic layers were washed with brine 15 mL(15 mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=3:1) to givecompound 54.3 (600 mg, 1.53 mmol, 90.69% yield) as a white solid. LCMS(ESI): m/z: [M+H] calcd for C₂₀H₂₉N₃O₅: 391; found 392; RT=0.681 min. ¹HNMR (400 MHz, chloroform-d) δ ppm 1.43-1.62 (m, 9H) 1.66-1.93 (m, 5H)2.49-2.62 (m, 1H) 3.02-3.12 (m, 1H) 3.14-3.25 (m, 1H) 3.71-3.87 (m, 2H)4.86-5 (m, 1H) 5.96 (br d, J=7.53 Hz, 1H) 6.72 (d, J=5.02 Hz, 1H) 7.77(s, 1H) δ (s, 1H) 8.16 (d, J=5.02 Hz, 1H).

To a solution of DIPA (426.51 mg, 4.21 mmol, 592.38 μL, 5.50 eq) in THF(6 mL) was added n-BuLi (2.5 M, 1.69 mL, 5.50 eq) at 0° C., the mixturewas stirred at 0° C. for 30 mins. The mixture was added to a solution ofcompound 54.3 (300 mg, 766.36 μmol, 1 eq) and chloroiodomethane (675.85mg, 3.83 mmol, 278.13 μL, 5 eq) in THF (6 mL) at −78° C. The mixture wasstirred at −78° C. for 30 mins. The reaction mixture was quenched byaddition of saturated aqueous NH₄Cl 5 mL, and then extracted with EtOAc15 mL (5 mL×3). The combined organic layers were washed with sat. aq.Na₂SO₃ 10 mL (10 mL×1), and brine 10 mL (10 mL×1), dried over Na₂SO₄,filtered and concentrated under reduced pressure to give compound 54.2(400 mg, crude) as a white solid.

To a mixture of compound 54.2 (400 mg, 975.82 μmol, 1 eq) and2,3,5,6-tetrafluorophenol (243.08 mg, 1.46 mmol, 1.50 eq) in DMF (5 mL)was added DIEA (504.46 mg, 3.90 mmol, 681.70 μL, 4 eq) in one portion at20° C. under N₂. The mixture was stirred at 20° C. for 10 hours. Theresidue was purified by prep-HPLC (neutral condition) to give compound54.1 (60 mg, 111.21 μmol, 11.40% yield) as a yellow solid. LCMS (ESI):m/z: [M+H] calcd for C₂₆H₂₉N₃O₅F₄: 539; found 540; RT=1.306 min.

To a solution of compound 54.1 (60 mg, 111.21 μmol, 1 eq) in DCM (10 mL)was added TFA (3.08 g, 27.01 mmol, 2 mL, 242.90 eq) in one portion at18° C. under N₂. The mixture was stirred at 18 for 15 hours. Thereaction mixture was concentrated under reduced pressure to give aresidue. The residue was purified by prep-HPLC (TFA condition) to giveProduct 54 (10 mg, 22.76 μmol, 20.46% yield) as a white solid. LCMS(ESI): m/z: [M+H] calcd for C₂₁H₂₁N₃O₃F₄: 439; found 440; RT=2.383 min.¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.40-1.52 (m, 3H) 1.57 (br d, J=6.53 Hz,3H) 1.63-1.76 (m, 2H) 2.78 (dd, J=13.61, 10.60 Hz, 1H) 3.13 (dd,J=13.61, 4.33 Hz, 1H) 4.65-4.74 (m, 1H) 5.22 (s, 2H) 6.71 (s, 1H) 6.75(d, J=6.78 Hz, 1H) 7.54-7.70 (m, 1H) 7.88 (br s, 3H) 8.34 (d, J=8.03 Hz,1H).

Example 55. Preparation ofN-(1-(2-aminopyridin-4-yl)-4-(2,6-difluorophenoxy)-3-oxobutan-2-yl)cyclopentanecarboxamide(55)

To a solution of compound 55.12 (58 g, 419.92 mmol, 1 eq) in MeOH (600mL) was added SOCl₂ (99.92 g, 839.84 mmol, 60.93 mL, 2 eq) dropwise at0° C. under N₂. The mixture was stirred at 0° C. for 30 mins, then washeated to 18° C. and stirred at 18° C. for 14.5 hours. The reactionmixture was concentrated under reduced pressure to give compound 55.11(65 g, crude) as a white solid. LCMS (ESI): m/z: [M+H] calcd forC₁H₈N₂O₂: 152; found 153; RT=0.100 min.

To a mixture of compound 55.11 (65 g, 427.21 mmol, 1 eq) and DMAP (2.61g, 21.36 mmol, 0.05 eq) in t-BuOH (500 mL) and ACETONE (150 mL) wasadded Boc₂O (279.72 g, 1.28 mol, 294.44 mL, 3 eq) dropwise at 18° C.under N₂. The mixture was stirred at 18° C. for 15 hours. The solutionwas diluted with pentane (200 ml), cooled in the refrigerator for 3hours and filtered to obtain compound 55.10 (110 g, 312.16 mmol, 73.07%yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C₁₇H₂₄N₂O₆:352; found 353; RT=0.877 min. ¹H NMR (400 MHz, chloroform-d) δ ppm1.39-1.50 (m, 19H) 3.97 (s, 3H) 7.77 (dd, J=5.02, 1.38 Hz, 1H) 7.82 (s,1H) 8.62 (d, J=5.02 Hz, 1H).

To a solution of compound 55.10 (60 g, 170.27 mmol, 1 eq) in THF (1 L)was added LiAlH4 (12.92 g, 340.54 mmol, 2 eq) portionwise at 0° C. underN₂. The mixture was stirred at 0° C. for 1 hour, then heated to 18° C.and stirred at 18° C. for 14 hours. The reaction mixture was quenched byaddition 8% NaOH (15 ml), filtered and then diluted with H₂O 1000 mL andextracted with EtOAc 1500 mL (500 mL×3). The combined organic layerswere washed with brine 1000 mL (1000 mL×1), dried over Na₂SO₄, filteredand concentrated under reduced pressure to give a residue. The residuewas purified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=2:1) to give compound 55.9 (15 g, 66.89 mmol, 39.28% yield) as awhite solid. LCMS (ESI): m/z: [M+H] calcd for C₁₁H₁₆N₂O₃: 293; found294; RT=0.313 min.

To a solution of compound 55.9 (8 g, 35.67 mmol, 1 eq) in DCM (60 mL)was added Dess-Martin periodinane (18.16 g, 42.81 mmol, 13.25 mL, 1.20eq) portionwise at 18° C. under N₂. The mixture was stirred at 18° C.for 2 hours. The reaction mixture was diluted with H₂O 60 mL andextracted with DCM 150 mL (50 mL×3). The combined organic layers werewashed with brine 100 mL (100 mL×1), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=5:1) to give compound 55.8 (5.10 g, 22.95 mmol, 64.33% yield) asa white solid. ¹H NMR (400 MHz, chloroform-d) δ ppm 1.59 (s, 9H) 7.41(dd, J=5.08, 1.32 Hz, 1H) 8.48 (s, 1H) 8.52 (d, J=5.15 Hz, 1H) 8.83 (brs, 1H).

To a solution of compound 55.7 (1.49 g, 4.50 mmol, 1 eq) in DCM (15 mL)was added DBU (1.03 g, 6.75 mmol, 1.02 mL, 1.50 eq) in one portion at18° C. under N₂. The mixture was stirred at 18° C. for 0.5 hour, andthen to the mixture was added compound 55.8 (1 g, 4.50 mmol, 1 eq) inone portion at 18° C., then was stirred at 18° C. for 0.5 hour. Thereaction mixture was concentrated under reduced pressure to give aresidue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1:1) to give compound 55.6 (1.90 g, 4.44mmol, 98.78% yield) as a white solid.

To a solution compound 55.6 (1 g, 2.34 mmol, 1 eq) in MeOH (50 mL) andTHF (50 mL) was added Pd—C (10%, 100 mg) under N₂. The suspension wasdegassed under vacuum and purged with H₂ several times. The mixture wasstirred under H₂ (50 psi) at 20° C. for 15 hours. The reaction mixturewas filtered and concentrated under reduced pressure to give compound55.5 (700 mg, crude) as a white solid. LCMS (ESI): m/z: [M+H] calcd forC₁₄H₁₉N₃O₄: 293; found 294; RT=0.638 min.

To a solution of compound 55.5 (600 mg, 2.05 mmol, 1 eq) in MeOH (50 mL)and THF (50 mL) was added Pd—C (10%, 100 mg) under N₂. The suspensionwas degassed under vacuum and purged with H₂ several times. The mixturewas stirred under H₂ (50 psi) at 20° C. for hours. The reaction mixturewas filtered and concentrated under reduced pressure to give compound55.4 (600 mg, crude) as a white solid. LCMS (ESI): m/z: [M+H] calcd forC₁₄H₂₁N₃O₄: 295; found 296; RT=0.239 min.

To a mixture of cyclopentanecarboxylic acid (193.24 mg, 1.69 mmol,184.04 μL, 1 eq) and EDCI (357.01 mg, 1.86 mmol, 1.10 eq) in DMF (10 mL)was added HOBt (251.64 mg, 1.86 mmol, 1.10 eq) in one portion at 0° C.under N₂. The mixture was stirred at 0° C. for 1 hour, then the mixturewas added dropwise a solution of compound 55.4 (500 mg, 1.69 mmol, 1 eq)in DMF (5 mL), then the mixture was added dropwise DIPEA (656.42 mg,5.08 mmol, 887.05 μL, 3 eq) and stirred at 0° C. for 1 hours. Thereaction mixture was diluted with H₂O 10 mL and extracted with EtOAc 15mL (5 mL×3). The combined organic layers were washed with brine 15 mL(15 mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=3:1) to givecompound 55.3 (600 mg, 1.53 mmol, 90.69% yield) as a white solid. LCMS(ESI): m/z: [M+H] calcd for C₂₀H₂₉N₃O₅: 391; found 392; RT=0.681 min. ¹HNMR (400 MHz, chloroform-d) δ ppm 1.43-1.62 (m, 9H) 1.66-1.93 (m, 5H)2.49-2.62 (m, 1H) 3.02-3.12 (m, 1H) 3.14-3.25 (m, 1H) 3.71-3.87 (m, 2H)4.86-5 (m, 1H) 5.96 (br d, J=7.53 Hz, 1H) 6.72 (d, J=5.02 Hz, 1H) 7.77(s, 1H) δ (s, 1H) 8.16 (d, J=5.02 Hz, 1H).

To a solution of DIPA (426.51 mg, 4.21 mmol, 592.38 μL, 5.50 eq) in THF(10 mL) was added n-BuLi (270.01 mg, 4.21 mmol, 5.50 eq) at 0° C., themixture was stirred at 0° C. for 30 mins. The mixture was added to asolution of compound 55.3 (300 mg, 766.36 μmol, 1 eq) andchloro(iodo)methane (675.85 mg, 3.83 mmol, 278.13 μL, 5 eq) in THF (10mL) at −78° C. The mixture was stirred at −78° C. for 30 mins. Thereaction mixture was quenched by addition aq sat NH₄Cl 5 mL, and thenextracted with EtOAc 15 mL (5 mL×3). The combined organic layers werewashed with aq sat Na₂SO₃ 10 mL (10 mL×1), and bine 10 mL (10 mL×1),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive compound 55.2 (400 mg, crude) as a white solid.

To a mixture of compound 55.2 (400 mg, 975.82 μmol, 1 eq) and2,6-difluorophenol (126.95 mg, 975.82 μmol, 1 eq) in DMF (5 mL) wasadded DIEA (504.46 mg, 3.90 mmol, 681.70 μL, 4 eq) in one portion at 20°C. under N₂. The mixture was stirred at 20° C. for 10 hours. The residuewas purified by prep-HPLC (neutral condition) to give compound 55.1 (60mg, 119.16 μmol, 12.21% yield) as a white solid. LCMS (ESI): m/z: [M+H]calcd for C₂₆H₃₁N₃O₅F₂: 503; found 504; RT=0.804 min.

To a solution of compound 55.1 (60 mg, 119.16 μmol, 1 eq) in DCM (3 mL)was added TFA (1.54 g, 13.51 mmol, 1 mL, 113.35 eq) in one portion at18° C. under N₂. The mixture was stirred at 18° C. for 15 hours. Thereaction mixture was concentrated under reduced pressure to give aresidue. The residue was purified by prep-HPLC (TFA condition) and SFC(condition: neutral condition) to give product 55 (10 mg, 24.79 μmol,25% yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd forC₂₁H₂₃N₃O₃F₂: 403; found 404; RT=2.518 min. ¹H NMR (400 MHz,METHANOL-d₄) δ ppm 1.46-1.58 (m, 3H) 1.58-1.71 (m, 3H) 1.71-1.85 (m, 2H)2.56-2.65 (m, 1H) 2.71-2.80 (m, 1H) 3.14 (dd, J=14, 5.40 Hz, 1H)4.85-5.02 (m, 2H) 6.41-6.55 (m, 2H) 6.93-7.03 (m, 2H) 7.03-7.11 (m, 1H)7.69-7.79 (m, 1H).

Examples 56, 57, and 58.(S)-N-(1-(6-aminopyridin-3-yl)-3-oxo-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)cyclopentanecarboxamide(56);(R)-N-(1-(6-aminopyridin-3-yl)-3-oxo-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)cyclopentanecarboxamide(57); and7V-(1-(6-aminopyridin-3-yl)-4-(2,6-difluorophenoxy)-3-oxobutan-2-yl)-2-methoxy-2-methylpropanamide(58)

Compounds 56, 57, and 58 were prepared according to the methodsdescribed above.

Compound No. Structure 56

57

58

Example 59. Preparation ofN-(1-(6-aminopyridin-3-yl)-4-(isoxazol-3-yloxy)-3-oxobutan-2-yl)-2-methoxy-2-methylpropanamide(59)

To a solution compound 59.9 (20 g, 167.89 mmol, 1 eq) in THF (500 mL)was added LiAlH₄ (12.74 g, 335.78 mmol, 2 eq) portions wise at 0° C.under N₂. The mixture was stirred at 0° C. for 2 hours. The reactionmixture was quenched by the addition of Na₂SO₄.10H₂O (30 g) thenfiltered. The filtrate was diluted with H₂O (200 mL) and extracted withEtOAc (200 mL×3). The combined organic layers were washed with brine(500 mL), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=3:1) to givecompound 59.8 (10 g, 81.88 mmol, 49% yield) as a yellow solid.

To a mixture of compound 59.8 (10 g, 81.88 mmol, 1 eq) and TEA (33.14 g,327.53 mmol, 45.59 mL, 4 eq) in THF (150 mL) was added Boc₂O (53.61 g,245.65 mmol, 56.43 mL, 3 eq) and DMAP (1.60 g, 13.10 mmol, 0.16 eq) inone portion at 25° C. under N₂. The mixture was stirred at 25° C. for 10hours. The reaction mixture was quenched by addition of Na₂SO₄.10H₂O (30g), then filtered. The filtrate was diluted with H₂O (200 mL) andextracted with EtOAc (200 mL×3). The combined organic layers were washedwith brine (500 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1) to givecompound 59.7 (11 g, 34.12 mmol, 42% yield) as a yellow solid. LCMS(ESI) m/z: [M+H]+ calcd for C₁₆H₂₃N₂O₅: 323; found 323; RT=1.537 min. ¹HNMR (400 MHz, chloroform-d) δ: 1.50 (s, 18H), 7.65 (d, J=8.33 Hz, 1H),8.18 (dd, 7=8.77, 2.19 Hz, 1H), 8.86 (d, 7=2.19 Hz, 1H), 10.02-10.10 (m,1H) ppm.

To a mixture of compound 59.7 (9.5 g, 29.47 mmol, 1 eq) and 59.6 (9.76g, 29.47 mmol, 1 eq) in DCM (60 mL) was added DBU (8.97 g, 58.94 mmol,8.88 mL, 2 eq) in one portion at 25° C. under N₂. The mixture wasstirred at 25° C. for 10 hours. The reaction mixture was quenched byaddition Na₂SO₄.10H₂O (30 g), then filtered, and then the filtrate wasdiluted with H₂O (200 mL) and extracted with EtOAc (200 mL×3). Thecombined organic layers were washed with brine (500 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1) to give compound 59.5 (7 g, 13.27mmol, 45% yield) as a yellow solid. LCMS (ESI) m/z: [M+H]+ calcd forC₂₇K₃₄N₃O₈: 528; found 528; RT=1.60 min.

To a solution of compound 59.5 (7 g, 13.27 mmol, 1 eq) in MeOH (200 mL)was added 10% Pd on carbon catalyst (800 mg) under N₂. The suspensionwas degassed under vacuum and purged with H₂ several times. The mixturewas stirred under H₂ (50 psi) at 25° C. for 10 hours. The reactionmixture was concentrated under reduced pressure to remove solvent. Theresidue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=3:1) to give compound 59.4 (2.8 g, 7.08 mmol, 53%yield) as a yellow solid. LCMS (ESI) m/z: [M+H]+ calcd for C₁₉H₃₀N₆O₃:396; found 396; RT=1.029 min.

To a mixture of compound 59.4 (836.43 mg, 7.08 mmol, 1 eq) and EDCI(1.63 g, 8.50 mmol, 1.2 eq) in DMF (30 mL) was added HOBt (1.15 g, 8.50mmol, 1.2 eq) in one portion at 0° C. under N₂. The mixture was stirredat 0° C. for 60 min, then the mixture was added2-methoxy-2-methylpropanoic acid (2.8 g, 7.08 mmol, 1 eq) and DIPEA(2.75 g, 21.24 mmol, 3.70 mL, 3 eq), then the mixture was stirred at 0°C. for 60 mins. The residue was diluted with H₂O (50 mL) and extractedwith EtOAc (50 mL×3). The combined organic layers were washed with H₂O(50 mL×3), The combined organic layers were washed with brine (100 mL),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=3:1) to give compound 59.3 (3 g, 6.05mmol, 86% yield) as yellow oil. LCMS (ESI): m/z: [M+H−Boc] calcd forC₁₉H₃₀N₃ O₆: 496; found 396; RT=1.516 min.

To a solution of DIPA (1.80 g, 17.76 mmol, 2.51 mL, 5.5 eq) in THF (50mL) was added n-BuLi (2.5 M, 7.10 mL, 5.5 eq) at 0° C., the mixture wasstirred at 0° C. for 30 mins. To the mixture was added a solution ofcompound 59.3 (1.6 g, 3.23 mmol, 1 eq) and chloroiodomethane (3.13 g,17.76 mmol, 1.29 mL, 5.5 eq) in THF (50 mL) at −78° C., then added DIPA(1.80 g, 17.76 mmol, 2.51 mL, 5.5 eq) at −78° C. The mixture was stirredat −78° C. for 3 h. The residue was diluted with H₂O (50 mL) andextracted with EtOAc (50 mL×3). The combined organic layers were washedwith H₂O (50 mL×3), The combined organic layers were washed with brine(100 mL), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=3:1) to givecompound 59.2 (100 mg, 194.55 μmol, 6% yield) as a white solid. LCMS(ESI) m/z: [M+H]+ calcd for C₂₄H₃₇N₃O₇ Cl: 514; found 514; RT=1.414 min.

To a mixture of compound 59.2 (100 mg, 194.55 μmol, 1 eq) andisoxazol-3-ol (16.55 mg, 194.55 μmol, 1 eq) in DMF (2 mL) was addedDIPEA (75.43 mg, 583.65 μmol, 101.66 μL, 3 eq) in one portion at 25° C.under N₂. The mixture was stirred at 25° C. for 10 hours. The residuewas diluted with H₂O (50 mL) and extracted with EtOAc (50 mL×3). Thecombined organic layers were washed with H₂O (50 mL×3); the combinedorganic layers were washed with brine (100 mL), dried over Na₂SO₄,filtered and concentrated under reduced pressure to give a residue. Theresidue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=3:1) to give compound 59.1 (150 mg) as a whitesolid. LCMS (ESI) m/z: [M+H-Boc] called for C₂₂H₃₁N₄O₇: 563; found 463;RT=0.937 min.

To a solution of compound 59.1 (300 mg, 533.23 μmol, 1 eq) in DCM (5 mL)was added TFA (533.23 μmol, 40 μL, 1 eq) in one portion at 25° C. underN₂. The mixture was stirred at 25° C. for 30 mins. The residue wasdiluted with H₂O (50 mL) and extracted with EtOAc (50 mL×3). Thecombined organic layers were washed with H₂O (50 mL×3) then washed withbrine (100 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=3:1) to give 59 (20mg) as a white solid. LCMS (ESI) m/z: [M+H]+ calcd for C₁₇H₂₃N₄O₅: 363;found 363; RT=2.357 min. ¹H NMR (400 MHz, METHANOL-d4) δ: 1.23 (s, 3H),1.33 (s, 3H), 2.84 (dd, J=14.55, 9.66 Hz, 1H), 3.21-3.28 (m, 4H),4.83-4.83 (m, 1H), 5.06 (d, 7=1.22 Hz, 2H), 6.18 (d, 7=1.83 Hz, 1H),6.92-6.98 (m, 1H), 7.67 (d, 7=1.47 Hz, 1H), 7.88 (dd, 7=9.17, 2.08 Hz,1H) and 8.39 (d, 7=1.83 Hz, 1H) ppm.

Example 60. Preparation ofN-(1-(6-aminopyridin-3-yl)-3-oxo-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)-2-methoxy-2-methylpropanamide(60)

To a solution of compound 60.9 (10 g, 83.95 mmol, 1 eq) in THF (300 mL)was added LAH (6.37 g, 167.9 mmol, 2 eq) at 0° C. The mixture wasstirred at 0° C. for 1.5 hour. The reaction mixture was quenched byaddition of saturated sodium sulfate at 0° C. and added 300 mL of H₂Othen extracted with ethyl acetate (300 mL×3). The combined organiclayers were washed with saturated brine (30 mL), dried over anhydroussodium sulfate, filtered and concentrated under reduced pressure to givecompound 60.8 (10 g, crude) as a yellow solid.

To a solution of compound 60.8 (10 g, 81.9 mmol, 1 eq) in THF (200 mL)was added TEA (33.14 g, 327.6 mmol, 45.4 mL, 4 eq), Boc₂O (53.62 g,245.67 mmol, 56.44 mL, 3 eq) and DMAP (2 g, 16.38 mmol, 0.2 eq). Themixture was stirred at 25° C. for 15 hours. The reaction mixture wasquenched by addition H₂O (300 mL) at 25° C. and extracted with ethylacetate (300 mL×3). The combined organic layers were washed withsaturated brine (50 mL), dried over anhydrous sodium sulfate, filteredand concentrated under reduced pressure to give a crude product. Theresidue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=2:1) to give compound 60.7 (4 g, 12.41 mmol, 15%yield) as a yellow solid. LCMS (ESI) m/z: [M+H]+ calcd for C₁₆H₂₂N₂O₅:323; found 167; RT=1.537 min.

To a solution of compound 60.7 (1 g, 3.1 mmol, 1 eq) in DCM (30 mL) wasadded DBU (944.53 mg, 6.20 mmol, 935.18 μL, 2 eq) and compound 60.6(1.03 g, 3.1 mmol, 1 eq). The mixture was stirred at 25° C. for 1 hour.The reaction mixture was quenched by addition H₂O (100 mL) at 25° C. andextracted with ethyl acetate (100 mL×3). The combined organic layerswere washed with saturated brines (5 mL), dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give acrude product. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1) to give compound 60.5 (2.6 g, 4.9mmol, 79% yield) as yellow oil. LCMS (ESI) m/z: [M+H]+ calcd forC₂₇H₃₃N₃O₈: 528; found 528; RT=1.612 min.

To a solution of compound 60.5 (2.6 g, 4.93 mmol, 1 eq) in MeOH (200 mL)was added 10% palladium on carbon (1 g) under N₂. The suspension wasdegassed under vacuum and purged with H₂ several times. The mixture wasstirred under H₂ (50 psi) at 25° C. for 15 hours. The reaction mixturewas filtered, and the filter was concentrated to give compound 60.4 (900mg, 2.28 mmol, 46% yield) as yellow oil. LCMS (ESI) m/z: [M+H]+ calcdfor C₁₉H₂₉N₃O₆: 396; found 396; RT=1.161 min.

To a solution of 2-methoxy-2-methylpropanoic acid (268.85 mg, 2.28 mmol,1 eq) in DMF (15 mL) was added HOBt (338.3 mg, 2.5 mmol, 1.1 eq) andEDCI (479.9 mg, 2.5 mmol, 1.1 eq). The mixture was stirred at 25° C. for1 hr. To this mixture was added compound 60.4 (900 mg, 2.28 mmol, 1 eq)and DIPEA (1.18 g, 9.1 mmol, 1.6 mL, 4 eq). The mixture was stirred at25° C. for 14 hours. The reaction mixture was quenched by addition H₂O(30 mL) at 25° C. and extracted with ethyl acetate (30 mL×3). Thecombined organic layers were washed with saturated brines (5 mL), driedover anhydrous sodium sulfate, filtered and concentrated under reducedpressure to give a crude product. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1) to givecompound 60.3 (960 mg, 1.94 mmol, 85% yield) as yellow oil. LCMS (ESI)m/z: [M+H]+ calcd for C₂₄H₃₇N₃O₈: 496; found 496; RT=1.487 min. ¹H NMR(400 MHz, chloroform-d) δ: 1.35 (d, J=8.38 Hz, 6H), 1.43 (s, 19H),3.06-3.31 (m, 5H), 3.74 (s, 3H), 4.72-4.97 (m, 1H), 7.09-7.21 (m, 2H),7.53 (dd, J=8.16, 2.21 Hz, 1H) and 8.24 (d, J=2.20 Hz, 1H) ppm.

To a solution of DIPA (842.3 mg, 8.32 mmol, 1.18 mL, 5.5 eq) in THF (5mL) was added n-BuLi (2.5 M, 3.33 mL, 5.5 eq) then was stirred at 0° C.for 0.5 hr under N₂. Then to the mixture was added to the solution ofcompound 60.3 (750 mg, 1.51 mmol, 1 eq) and chloroiodomethane (1.47 g,8.32 mmol, 604.19 μL, 5.5 eq) in THF (15 mL) was stirred at −78° C. for2.5 hours. The reaction mixture was quenched by addition of saturatedNH₄Cl (20 mL) at 25° C. and extracted with ethyl acetate (15 mL×3). Thecombined organic layers were washed with saturated Na₂SO₃ (10 mL) andsaturated NaHCO₃ (10 mL) and saturated brine (10 mL), dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to give a crude product. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1) to givecompound 60.2 (50 mg, 97.3 μmol, 6% yield) as yellow oil. LCMS (ESI)m/z: [M+H]+ calcd for C₂₄H₃₆N₃O₇Cl: 514; found 414; RT=1.253 min.

To a solution of compound 60.2 (40 mg, 77.8 μmol, 1 eq) in DMF (2 mL)was added DIEA (30.17 mg, 233.5 μmol, 40.7 μL, 3 eq) and2,3,5,6-tetrafluorophenol (19.4 mg, 116.73 μmol, 1.5 eq). The mixturewas stirred at 25° C. for 12 hours. The reaction mixture was quenched byaddition H₂O (10) mL at 25° C. and extracted with ethyl acetate (10mL×3). The combined organic layers were washed with saturated brine (5mL), dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure to give a crude product. The residue was purifiedby column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1:1) togive compound 60.1 (20 mg, 31.07 μmol, 40% yield) as a white solid. LCMS(ESI) m/z: [M+H]+ calcd for C₃₀H₃₇N₃O₈F₄: 644; found 544; RT=1.402 min.

A mixture of compound 60.1 (20 mg, 31.07 μmol, 1 eq) in TFA (1 mL) andDCM (5 mL) was stirred at 25° C. for 15 hours. The mixture wasconcentrated under reduced pressure to give a residue. The residue waspurified by prep-HPLC (neutral condition) to give 60 (10 mg, 22.55 μmol,73% yield) as a white solid. LCMS (ESI) m/z: [M+H]+⁺ calcd forC₂₀H₂₁N₃O₄F₄: 444; found 444; RT=1.68 min. ¹H NMR (400 MHz, METHANOL-d₄)δ: 1.15-1.33 (m, 6H), 2.72-2.90 (m, 1H), 3.04-3.23 (m, 4H), 4.94-5 (m,1H), 5.02-5.25 (m, 2H), 6.53 (br d, J=8.16 Hz, 1H), 7.12 (br s, 1H),7.37 (br d, J=7.50 Hz, 1H) and 7.65-7.78 (m, 1H) ppm.

Example 61. Preparation ofN-(1-(6-aminopyridin-3-yl)-3-oxo-4-(2,3,6-trifluorophenoxy)butan-2-yl)-2-methoxy-2-methylpropanamide(61)

To a solution of compound 60.2 (50 mg, 97.3 μmol, 1 eq) in DMF (3 mL)was added DIEA (37.72 mg, 291.82 μmol, 50.83 μL, 3 eq) and2,3,6-trifluorophenol (28.81 mg, 194.55 μmol, 2 eq). The mixture wasstirred at 25° C. for 15 hours. The reaction mixture was quenched byaddition H₂O (10 mL) at 25° C. and extracted with ethyl acetate (10mL×3). The combined organic layers were washed with saturated brine (5mL), dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure to give a crude product. The residue was purifiedby column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1:1) togive compound 61.1 (15 mg, 24 μmol, 25% yield) as yellow oil. LCMS (ESI)m/z: [M+H]+ calcd for C₃₀H₃₈N₃O₈F₈: 626; found 526; RT=1.389 min.

A mixture of compound 61.1 (40 mg, 63.94 μmol, 1 eq) in TFA (1 mL) andDCM (5 mL) was stirred at 25° C. for 1 hr. The mixture was concentratedunder reduced pressure to give a residue. The residue was purified bysemi-preparative scale HPLC (TFA condition) to give 61 (3 mg, 7.1 μmol,11% yield) as a yellow solid. LCMS (ESI) m/z: [M+H]+ calcd forC₂₀H₂₂N₃O₄F₃: 426; found 426; RT=1.664 min. ¹H NMR (400 MHz, DMSO-d₆) δ:1.10 (s, 3H), 1.21 (s, 3H), 2.69-2.79 (m, 1H), 2.94 (br dd, J=14.06,4.16 Hz, 1H), 3.01 (s, 3H), 4.47-4.65 (m, 1H), 4.98-5.30 (m, 2H), 5.70(s, 2H), 6.32 (d, J=8.31 Hz, 1H), 7.19 (td, J=9.35, 6.72 Hz, 3H), 7.71(s, 1H) and 8.07 (br d, J=8.31 Hz, 1H) ppm.

Example 62. Preparation ofN-(1-(6-aminopyridin-3-yl)-3-oxo-4-(2,3,6-trifluorophenoxy)butan-2-yl)-2-methoxy-2-methylpropanamide(62)

To a mixture of compound 62.7 (1.4 g, 6.3 mmol, 1 eq) and compound 62.6(2.09 g, 6.3 mmol, 1 eq) in DCM (30 mL) was added DBU (1.92 g, 12.6mmol, 1.9 mL, 2 eq) in one portion at 18° C. under N₂. The mixture wasstirred at 18° C. for 1 hour. The reaction mixture was concentratedunder reduced pressure to give a residue. This was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1) to givecompound 62.5 (1.2 g, 2.81 mmol, 44.6% yield) as a white solid.

To a solution of compound 62.5 (1 g, 2.34 mmol, 1 eq) in MeOH (20 mL)was added Pd—C (10% palladium on carbon, 100 mg) under N₂. Thesuspension was degassed under vacuum and purged with H₂ several times.The mixture was stirred under H₂ (50 psi) at 25° C. for 10 hours. Thereaction mixture was filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Ethyl acetate) to give compound 62.4 (270 mg, 914.2 μmol, 39.1% yield)as a white solid.

To a mixture of 2-methoxy-2-methylpropanoic acid (129.6 mg, 1.1 mmol,1.2 eq) and EDCI (210.31 mg, 1.1 mmol, 1.2 eq) in DMF (5 mL) was addedHOBt (148.24 mg, 1.1 mmol, 1.2 eq) in one portion at 18° C. under N₂.The mixture was stirred at 18° C. for 30 mins, then compound 62.4 (270mg, 914.22 μmol, 1 eq) and DIPEA (354.47 mg, 2.74 mmol, 477.7 μL, 3 eq)were added in one portion, the mixture was stirred at 18° C. for 30mins. The reaction mixture was diluted with H₂O (5 mL) and extractedwith EtOAc (3 mL×3). The combined organic layers were washed with brine(10 mL), dried over Na₂SO₄, filtered and concentrated under reducedpressure. This residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1) to give compound 62.3 (250 mg, 632.2μmol, 69.2% yield) as a white solid. LCMS (ESI) m/z: [M+H]+ calcd. forC₁₉H₂₉N₃O₆: 395; found 396; RT=0.88 min. ¹H NMR (400 MHz, DMSO-d6) δ:1.12 (s, 3H), 1.20 (s, 3H), 1.47 (s, 9H), 3.05 (s, 3H), 3.06-3.15 (m,2H), 3.66 (s, 3H), 4.49-4.61 (m, 1H), 6.90 (dd, J=5.07, 1.28 Hz, 1H),7.71 (s, 1H), 8.02-8.16 (m, 2H) and 9.68 (s, 1H) ppm.

To a solution of DIPA (351.8 mg, 3.5 mmol, 491.4 μL, 5.5 eq) in THF (5mL) was added n-BuLi (2.5 M, 1.4 mL, 5.5 eq) at 0° C., the mixture wasstirred at 0° C. for 30 mins. To the mixture was added a solution ofcompound 62.3 (250 mg, 632.2 μmol, 1 eq) and chloroiodomethane (613.3mg, 3.48 mmol, 252.4 μL, 5.5 eq) in THF (5 mL) at −78° C. The mixturewas stirred at −78° C. for 30 minutes. The reaction mixture was quenchedby addition of aqueous saturated NH₄Cl (5 mL) and extracted with EtOAc(2 mL×3). The combined organic layers were washed with aqueous saturatedNa₂SO₃ (3 mL) and brine (5 mL), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give compound 62.2 (400 mg,crude) as yellow oil. LCMS (ESI) m/z: [M+H]+ calcd. for C₁₉H₂₈N₃O₅Cl:413; found 414; RT=1.2 min.

To a mixture of compound 62.2 (400 mg, 966.4 μmol, 1 eq) and2,6-difluorophenol (125.7 mg, 966.4 μmol, 1 eq) in DMF (2 mL) was addedDIEA (374.71 mg, 2.9 mmol, 505 μL, 3 eq) in one portion at 18° C. underN₂. The mixture was stirred at 18° C. for 10 hours. The residue waspurified by prep-HPLC (neutral condition) to give compound 62.1 (20 mg,39.4 μmol, 4.1% yield) as a white solid. LCMS (ESI) m/z: [M+H]+ calcdfor C₂₅H₃₁N₃O₆F₂: 507; found 508; RT=1.076 min.

To a solution of compound 62.1 (20 mg, 39.41 μmol, 1 eq) in DCM (5 mL)was added TFA (1 mL) in one portion at 18° C. under N₂. The mixture wasstirred at 18° C. for 10 hours. The reaction mixture was concentratedunder reduced pressure to give 62 (8 mg, 19.6 μmol, 50% yield) as ayellow solid. LCMS (ESI) m/z: [M+H]+ calcd. for C₂₀H₂₃N₃O₄F₂: 407; found408; RT=2.41 min. ¹H NMR (400 MHz, DMSO-d6) δ: 1.11 (s, 3H), 1.21 (s,3H), 2.84-2.94 (m, 1H), 3.04 (s, 3H), 3.16-3.24 (m, 1H), 4.75-4.85 (m,1H), 4.97-5.16 (m, 2H), 6.74 (s, 1H), 6.79 (d, J=6.58 Hz, 1H), 7.05-7.18(m, 3H), 7.85 (d, J=6.58 Hz, 1H), 8.04 (br s, 2H) and 8.35 (d, J=8.77Hz, 1H) ppm.

Example 63. Preparation ofN-(1-(2-aminopyridin-4-yl)-4-(isoxazol-3-yloxy)-3-oxobutan-2-yl)-2-methoxy-2-methylpropanamide(63)

To a solution of compound 63.11 (30 g, 217.2 mmol, 1 eq) in MeOH (500mL) was added SOCl₂ (103.4 g, 868.8 mmol, 63 mL, 4 eq) dropwise at 0° C.under N₂, then it was stirred at 18° C. for 10 hours. The reactionmixture was concentrated under reduced pressure to give compound 63.10(33 g, crude) as a white solid. LCMS (ESI): m/z: [M+H]+ calcd forC₇H₉N₂O₂: 153; found 153; RT=0.3 min.

To a mixture of compound 63.10 (10 g, 65.72 mmol, 1 eq) and DMAP (401.48mg, 3.29 mmol, 0.05 eq) in t-BuOH (500 mL) and ACETONE (150 mL) wasadded Boc₂O (43 g, 197.2 mmol, 45.30 mL, 3 eq) dropwise at 18° C. underN₂. The mixture was stirred at 18° C. for hours. The solution wasdiluted with pentane (200 mL), cooled in the refrigerator for 3 hoursthen filtered to give compound 63.9 (25 g, 99.1 mmol, 50.3% yield) as awhite solid. ¹H NMR (400 MHz, METHANOL-d4) δ: 1.48 (s, 9H), 3.80-3.96(m, 3H), 7.44 (dd, J=5.07, 1.41 Hz, 1H), 8.33 (s, 1H), 8.43 (d, J=5.01Hz, 1H) and 10.11 (s, 1H) ppm.

To a solution of compound 63.9 (25 g, 99.1 mmol, 1 eq) in THF (500 mL)was added LiAlH₄ (7.52 g, 198.2 mmol, 2 eq) portion-wise at 0° C. underN₂. The mixture was stirred at 0° C. for 1 hour, then it was stirred at18° C. for 14 hours. The reaction mixture was quenched by addition 8%NaOH (15 mL), filtered and then diluted with H₂O (1000 mL) and extractedwith EtOAc (500 mL×3). The combined organic layers were washed withbrine (1000 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure. The residue was purified by column chromatography(SiO₂, Petroleum ether/Ethyl acetate=2:1) to give compound 63.8 (7 g,31.21 mmol, 31.5% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ:1.47 (s, 8H), 4.50 (d, J=5.73 Hz, 2H), 5.40 (t, J=5.73 Hz, 1H), 6.94(dd, J=5.07, 0.66 Hz, 1H), 7.81 (s, 1H), 8.15 (d, J=5.07 Hz, 1H) and9.70 (br s, 1H) ppm.

To a solution of compound 63.8 (7 g, 31.2 mmol, 1 eq) in DCM (60 mL) wasadded Dess-Martin periodinane (19.9 g, 46.8 mmol, 14.5 mL, 1.5 eq)portion-wise at 18° C. under N₂, then the mixture was stirred at 18° C.for 2 hours. The reaction mixture was diluted with H₂O (60 mL) andextracted with DCM (50 mL×3). The combined organic layers were washedwith brine (100 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure to give a residue. This residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=5:1) to givecompound 63.7 (5.8 g, 26.1 mmol, 83.6% yield) as a white solid. ¹H NMR(400 MHz, DMSO-d6) δ: 1.49 (s, 9H), 7.42 (dd, J=4.96, 0.99 Hz, 1H), 8.25(s, 1H), 8.50 (d, J=4.85 Hz, 1H), 10.04 (s, 1H) and 10.12 (s, 1H) ppm.

To a mixture of compound 63.7 (5.8 g, 26.1 mmol, 1 eq) and compound 63.6(8.65 g, 26.1 mmol, 1 eq) in DCM (60 mL) was added DBU (7.95 g, 52.2mmol, 7.87 mL, 2 eq) in one portion at 18° C. under N₂. The mixture wasstirred at 18° C. for 10 hours. The reaction mixture was concentratedunder reduced pressure to give a residue, then it was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1) to givecompound 63.5 (3 g, 7 mmol, 27% yield) as a white solid. LCMS (ESI) m/z:[M+H]+ calcd for C₂₂H₂₆N₃O₆: 428; found 428; RT=1.34 min.

To a solution of compound 63.5 (3 g, 7 mmol, 1 eq) in MeOH (20 mL) wasadded Pd—C (10% palladium on carbon, 100 mg) under N₂. The suspensionwas degassed under vacuum and purged with H₂ several times. The mixturewas stirred under H₂ (50 psi) at 25° C. for 10 hours. The reactionmixture was filtered and concentrated under reduced pressure to give aresidue. The residue was purified by column chromatography (SiO₂, Ethylacetate) to give compound 63.4 (1.32 g, 4.47 mmol, 64% yield) as a whitesolid. LCMS (ESI) m/z: [M+H]+ calcd for C₁₄H₂₂N₃O₄: 296; found 296;RT=0.694 min. ¹H NMR (400 MHz, DMSO-d6) δ: 1.46 (s, 9H), 2.69-2.79 (m,1H), 2.81-2.91 (m, 1H), 3.54-3.62 (m, 4H), 6.85 (d, J=4.82 Hz, 1H), 7.64(s, 1H), 8.10 (d, J=4.82 Hz, 1H) and 9.71 (s, 1H) ppm.

To a mixture of 2-methoxy-2-methylpropanoic acid (633.6 mg, 5.36 mmol,1.2 eq) and EDCI (1.03 g, 5.36 mmol, 1.2 eq) in DMF (15 mL) was addedHOBt (724.72 mg, 5.36 mmol, 1.2 eq) in one portion at 18° C. under N2.The mixture was stirred at 18° C. for 30 mins, then was added compound63.4 (1.32 g, 4.47 mmol, 1 eq) and DIPEA (1.73 g, 13.41 mmol, 2.34 mL, 3eq) in one portion, the mixture was stirred at 18° C. for 30 mins. Thereaction mixture was diluted with H₂O (10 mL) and extracted with EtOAc(10 mL×3). The combined organic layers were washed with brine (10 mL),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1) to give compound 63.3 (1.5 g, 3.79mmol, 85% yield) as a white solid. LCMS (ESI) m/z: [M+H]+ calcd forC₁₉H₃₀N₃O₆: 396; found 396; RT=0.892 min. ¹H NMR (400 MHz, METHANOL-d4)δ: 1.31 (s, 3H), 1.37 (s, 3H), 1.56-1.64 (m, 9H), 3.16 (dd, J=13.89,9.26 Hz, 1H), 3.25 (s, 3H), 3.34 (dd, J=13.89, 4.85 Hz, 1H), 3.84 (s,3H), 4.82 (dd, J=9.15, 4.96 Hz, 1H), 6.96 (dd, J=5.07, 1.32 Hz, 1H),7.78-7.89 (m, 1H) and 8.18 (d, J=5.07 Hz, 1H) ppm.

To a solution of DIPA (281.48 mg, 2.78 mmol, 393.12 μL, 5.5 eq) in THF(5 mL) was added n-BuLi (2.5 M, 1.11 mL, 5.5 eq) at 0° C., the mixturewas stirred at 0° C. for 30 mins. The mixture was added a solution ofcompound 63.3 (0.2 g, 505.75 μmol, 1 eq) and chloroiodomethane (490.64mg, 2.78 mmol, 201.9 μL, 5.5 eq) in THF (5 mL) at −78° C. The mixturewas stirred at −78° C. for 30 mins. The reaction mixture was quenched byaddition aqueous saturated NH₄Cl (5 mL) and then extracted with EtOAc (5mL×3). The combined organic layers were washed with aqueous saturatedNa₂SO₃ (10 mL), and brine (50 mL), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give compound 63.2 (200 mg,crude) as yellow oil.

To a mixture of compound 63.2 (100 mg, 241.6 μmol, 1 eq) andisoxazol-3-ol (20.55 mg, 241.6 μmol, 1 eq) in DMF (2 mL) was added DIEA(93.7 mg, 724.8 μmol, 126.25 μL, 3 eq) in one portion at 18° C. underN₂. The mixture was stirred at 18° C. for 10 hours. The reaction mixturewas diluted with H₂O (2 mL) and extracted with EtOAc (2 mL×3). Thecombined organic layers were washed with brine (5 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1:1) to give compound 63.1 (20 mg, 43.24μmol, 18% yield) as a white solid. LCMS (ESI) m/z: [M+H]+ calcd forC₂₂H₃₁N₄O₇: 463; found 463; RT=1.203 min.

To a solution of compound 63.1 (20 mg, 43.24 μmol, 1 eq) in DCM (5 mL)was added TFA (1 mL) in one portion at 18° C. under N₂. The mixture wasstirred at 18° C. for 10 mins. The reaction mixture was concentratedunder reduced pressure to give a residue. The residue was purified byprep-HPLC (TFA condition) to give 63 (5 mg, 13.8 μmol, 32% yield) as awhite solid. LCMS (ESI) m/z: [M+H]+ calcd for C₁₇H₂₃N₄O₅: 363; found363; RT=2.688 min. ¹H NMR (400 MHz, METHANOL-d4) δ: 1.24 (s, 3H), 1.33(s, 3H), 2.96 (dd, J=14.11, 10.14 Hz, 1H), 3.23 (s, 3H), 3.37 (dd,J=14.11, 4.63 Hz, 1H), 4.95 (dd, J=9.92, 4.63 Hz, 1H), 5.06 (s, 2H),6.18 (d, J=1.54 Hz, 1H), 6.82-6.86 (m, 2H), 7.74 (d, J=7.06 Hz, 1H) and8.39 (d, J=1.76 Hz, 1H) ppm.

Example 64. Preparation ofN-(1-(2-aminopyridin-4-yl)-3-oxo-4-(2,3,6-trifluorophenoxy)butan-2-yl)-2-methoxy-2-methylpropanamide(64)

To a solution of compound 64.11 (30 g, 217.2 mmol, 1 eq) in MeOH (500mL) was added SOCl₂ (103.4 g, 868.8 mmol, 63 mL, 4 eq) dropwise at 0° C.under N₂, then heated to 18° C. and stirred for 10 hours. The reactionmixture was concentrated under reduced pressure to give compound 64.10(33 g, crude) as a white solid. LCMS (ESI): m/z: [M+H]+ calcd forC₇H₉N₂O₂: 153; found 153; RT=0.3 min.

To a mixture of compound 64.10 (10 g, 65.72 mmol, 1 eq) and DMAP (401.48mg, 3.29 mmol, 0.05 eq) in t-BuOH (500 mL) and ACETONE (150 mL) wasadded Boc₂O (43.03 g, 197.17 mmol, 45.3 mL, 3 eq) dropwise at 18° C.under N₂. The mixture was stirred at 18° C. for hours. The solution wasdiluted with pentane (200 mL), cooled in the refrigerator for 3 hoursand filtered to give compound 64.9 (25 g, 99.1 mmol, 50% yield) as awhite solid. ¹H NMR (400 MHz, METHANOL-d4) δ: 1.48 (s, 9H), 3.80-3.96(m, 3H), 7.44 (dd, J=5.07, 1.41 Hz, 1H), 8.33 (s, 1H), 8.43 (d, J=5.01Hz, 1H) and 10.11 (s, 1H) ppm.

To a solution of compound 64.9 (25 g, 99.1 mmol, 1 eq) in THF (500 mL)was added LiAlH₄ (7.52 g, 198.2 mmol, 2 eq) portion-wise at 0° C. underN₂. The mixture was stirred at 0° C. for 1 hour, then heated to 18° C.and stirred at 18° C. for 14 hours. The reaction mixture was quenched byaddition 8% NaOH (15 mL), filtered and then diluted with H₂O (1000 mL)and extracted with EtOAc (500 mL×3). The combined organic layers werewashed with brine (1000 mL), dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1) to givecompound 64.8 (7 g, 31.21 mmol, 32% yield) as a white solid. ¹H NMR (400MHz, DMSO-d6) δ: 1.47 (s, 8H), 4.50 (d, J=5.73 Hz, 2H), 5.40 (t, J=5.73Hz, 1H), 6.94 (dd, J=5.07, 0.66 Hz, 1H), 7.81 (s, 1H), 8.15 (d, J=5.07Hz, 1H) and 9.70 (br s, 1H) ppm.

To a solution of compound 64.8 (7 g, 31.21 mmol, 1 eq) in DCM (60 mL)was added Dess-Martin periodinane (19.9 g, 46.8 mmol, 14.5 mL, 1.5 eq)portion-wise at 18° C. under N₂. The mixture was stirred at 18° C. for 2hours. The reaction mixture was diluted with H₂O (60 mL) and extractedwith DCM (50 mL×3). The combined organic layers were washed with brine(100 mL×1), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=5:1) to givecompound 64.7 (5.8 g, 26.1 mmol, 84% yield) as a white solid. ¹H NMR(400 MHz, DMSO-d6) δ: 1.49 (s, 9H), 7.42 (dd, J=4.96, 0.99 Hz, 1H), 8.25(s, 1H), 8.50 (d, J=4.85 Hz, 1H), 10.04 (s, 1H) and 10.12 (s, 1H) ppm.

To a mixture of compound 64.7 (5.8 g, 26.1 mmol, 1 eq) and compound 64.6(8.65 g, 26.1 mmol, 1 eq) in DCM (60 mL) was added DBU (7.95 g, 52.20mmol, 7.87 mL, 2 eq) in one portion at 18° C. under N₂. The mixture wasstirred at 18° C. for 10 hours. The reaction mixture was concentratedunder reduced pressure to give a residue. The residue was purified bycolumn chromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1) to givecompound 64.5 (3 g, 7.02 mmol, 27% yield) as a white solid. LCMS (ESI)m/z: [M+H]+ calcd for C₂₂H₂₆N₃O₆: 428; found 428; RT=1.343 min.

To a solution of compound 64.5 (3 g, 7.02 mmol, 1 eq) in MeOH (20 mL)was added 10% palladium on carbon catalyst (100 mg) under N₂. Thesuspension was degassed under vacuum and purged with H₂ several times.The mixture was stirred under H₂ (50 psi) at 25° C. for hours. Thereaction mixture was filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Ethyl acetate) to give compound 64.4 (1.32 g, 4.47 mmol, 64% yield) as awhite solid. LCMS (ESI) m/z: [M+H]+ calcd for C₁₄H₂₂N₃O₄: 296; found296; RT=0.694 min. ¹H NMR (400 MHz, DMSO-d6) δ: 1.46 (s, 9H), 2.69-2.79(m, 1H), 2.81-2.91 (m, 1H), 3.54-3.62 (m, 4H), 6.85 (d, J=4.82 Hz, 1H),7.64 (s, 1H), 8.10 (d, J=4.82 Hz, 1H) and 9.71 (s, 1H) ppm.

To a mixture of 2-methoxy-2-methylpropanoic acid (633.59 mg, 5.36 mmol,1.2 eq) and EDCI (1.03 g, 5.36 mmol, 1.2 eq) in DMF (15 mL) was addedHOBt (724.72 mg, 5.36 mmol, 1.2 eq) in one portion at 18° C. under N₂.The mixture was stirred at 18° C. for 30 mins, and then was addedcompound 64.4 (1.32 g, 4.47 mmol, 1 eq) and DIPEA (1.73 g, 13.41 mmol,2.34 mL, 3 eq) in one portion, the mixture was stirred at 18° C. for 30mins. The reaction mixture was diluted with H₂O (10 mL) and extractedwith EtOAc (10 mL×3). The combined organic layers were washed with brine(10 mL), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1) to givecompound 64.3 (1.5 g, 3.8 mmol, 85% yield) as a white solid. LCMS (ESI)m/z: [M+H]+ calcd for C₁₉H₃₀N₃O₆: 396; found 396; RT=0.892 min. ¹H NMR(400 MHz, METHANOL-d4) δ: 1.31 (s, 3H), 1.37 (s, 3H), 1.56-1.64 (m, 9H),3.16 (dd, J=13.89, 9.26 Hz, 1H), 3.25 (s, 3H), 3.34 (dd, J=13.89, 4.85Hz, 1H), 3.84 (s, 3H), 4.82 (dd, J=9.15, 4.96 Hz, 1H), 6.96 (dd, J=5.07,1.32 Hz, 1H), 7.78-7.89 (m, 1H) and 8.18 (d, J=5.07 Hz, 1H) ppm.

To a solution of DIPA (1.41 g, 13.91 mmol, 1.97 mL, 5.5 eq) in THF (20mL) was added n-BuLi (2.5 M, 5.56 mL, 5.5 eq) at 0° C., the mixture wasstirred at 0° C. for 30 mins. The mixture was added a solution ofcompound 64.3 (1 g, 2.53 mmol, 1 eq) and chloroiodomethane (2.45 g,13.91 mmol, 1.01 mL, 5.5 eq) in THF (20 mL) at −78° C. The mixture wasstirred at −78° C. for 30 mins. The reaction mixture was quenched byaddition aqueous saturated NH₄Cl (20 mL) and then extracted with EtOAc(20 mL×3). The combined organic layers were washed with aqueoussaturated Na₂SO₃ (30 mL), and brine (50 mL), dried over Na₂SO₄, filteredand concentrated under reduced pressure to give compound 64.2 (2 g) asyellow oil.

To a mixture of compound 64.2 (650 mg, 1.57 mmol, 1 eq) and2,3,6-trifluorophenol (232.49 mg, 1.57 mmol, 1 eq) in DMF (2 mL) wasadded DIEA (608.89 mg, 4.71 mmol, 820.61 μL, 3 eq) in one portion at 18°C. under N₂. The mixture was stirred at 18° C. for 10 hours. The residuewas purified by prep-HPLC (TFA condition) to give compound 64.1 (30 mg,57.1 μmol, 4% yield) as a white solid. LCMS (ESI) m/z: [M+H]+ calcd forC₂₅H₃₁N₃O₆F₃: 526; found 526; RT=1.353 min. ¹H NMR (400 MHz,METHANOL-d4) δ: 1.22 (s, 3H), 1.31 (s, 2H), 1.56-1.61 (m, 9H), 3.06 (dd,J=14.11, 9.92 Hz, 1H), 3.20 (s, 2H), 3.43-3.53 (m, 1H), 5-5.13 (m, 2H),6.94-7.07 (m, 3H), 7.21-7.33 (m, 2H) and 8.11-8.20 (m, 1H) ppm.

To a solution of compound 64.1 (30 mg, 57.1 μmol, 1 eq) in DCM (5 mL)was added TFA (1 mL) in one portion at 18° C. under N₂. The mixture wasstirred at 18° C. for 10 mins. The residue was purified by prep-HPLC(TFA condition) to give 64 (10 mg, 23.51 μmol, 41% yield) as a whitesolid. LCMS (ESI) m/z: [M+H]+ calcd for C₂₀H₂₃N₃O₄F₃: 426; found 426;RT=2.646 min. ¹H NMR (400 MHz, DMSO-d6) δ: 1.15 (s, 3H), 1.22 (s, 3H),2.91 (dd, J=14, 10.03 Hz, 1H), 3.07 (s, 3H), 3.19 (dd, J=14, 4.30 Hz,1H), 4.77 (td, J=9.32, 4.52 Hz, 1H), 5.09 (q, J=17.35 Hz, 2H), 6.71 (brd, J=4.19 Hz, 2H), 7.08-7.16 (m, 2H), 7.78-7.82 (m, 1H) and 8.07 (br d,J=8.82 Hz, 1H) ppm.

Example 65. Preparation ofN-(1-(2-aminopyridin-4-yl)-3-oxo-4-(2,3,5,6-tetrafluorophenoxy)butan-2-yl)-2-methoxy-2-methylpropanamide(65)

To a solution of compound 65.11 (30 g, 217.2 mmol, 1 eq) in MeOH (500mL) was added SOCl₂ (103.36 g, 868.8 mmol, 63.02 mL, 4 eq) dropwise at0° C. under N₂, then heated to 18° C. and stirred for 10 hours. Thereaction mixture was concentrated under reduced pressure to givecompound 65.10 (33 g, crude) as a white solid. LCMS (ESI) m/z: [M+H]+calcd for C₇H₉N₂O₂: 153; found 153; RT=0.298 min.

To a mixture of compound 65.10 (10 g, 65.7 mmol, 1 eq) and DMAP (401.5mg, 3.29 mmol, 0.05 eq) in t-BuOH (500 mL) and ACETONE (150 mL) wasadded Boc₂O (43.03 g, 197.17 mmol, 45.3 mL, 3 eq) dropwise at 18° C.under N₂. The mixture was stirred at 18° C. for 15 hours. The solutionwas diluted with pentane (200 mL), cooled in the refrigerator for 3hours and filtered to give compound 65.9 (25 g, 99.1 mmol, 50% yield) asa white solid. ¹H NMR (400 MHz, METHANOL-d4) δ: 1.48 (s, 9H), 3.80-3.96(m, 3H), 7.44 (dd, J=5.07, 1.41 Hz, 1H), 8.33 (s, 1H), 8.43 (d, J=5.01Hz, 1H) and 10.11 (s, 1H) ppm.

To a solution of compound 65.9 (25 g, 99.1 mmol, 1 eq) in THF (500 mL)was added LiAlH₄ (7.52 g, 198.2 mmol, 2 eq) portion-wise at 0° C. underN₂. The mixture was stirred at 0° C. for 1 hour, then heated to 18° C.and stirred at 18° C. for 14 hours. The reaction mixture was quenched byaddition 8% NaOH (15 mL), filtered and then diluted with H₂O (1000 mL)and extracted with EtOAc (500 mL×3). The combined organic layers werewashed with brine (1000 mL), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=2:1) to give compound 65.8 (7 g, 31.2 mmol, 32% yield) as awhite solid. ¹H NMR (400 MHz, DMSO-d6) δ: 1.47 (s, 8H), 4.50 (d, J=5.73Hz, 2H), 5.40 (t, J=5.73 Hz, 1H), 6.94 (dd, J=5.07, 0.66 Hz, 1H), 7.81(s, 1H), 8.15 (d, J=5.07 Hz, 1H) and 9.70 (br s, 1H) ppm.

To a solution of compound 65.8 (7 g, 31.21 mmol, 1 eq) in DCM (60 mL)was added Dess-Martin periodinane (19.86 g, 46.82 mmol, 14.50 mL, 1.5eq) portion-wise at 18° C. under N₂. The mixture was stirred at 18° C.for 2 hours. The reaction mixture was diluted with H₂O (60 mL) andextracted with DCM (50 mL×3). The combined organic layers were washedwith brine (100 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=5:1) to givecompound 65.7 (5.8 g, 26.1 mmol, 84% yield) as a white solid. ¹H NMR(400 MHz, DMSO-d6) δ: 1.49 (s, 9H), 7.42 (dd, J=4.96, 0.99 Hz, 1H), 8.25(s, 1H), 8.50 (d, J=4.85 Hz, 1H), 10.04 (s, 1H) and 10.12 (s, 1H) ppm.

To a mixture of compound 65.7 (5.8 g, 26.1 mmol, 1 eq) and compound 65.6(8.65 g, 26.1 mmol, 1 eq) in DCM (60 mL) was added DBU (7.95 g, 52.2mmol, 7.87 mL, 2 eq) in one portion at 18° C. under N₂. The mixture wasstirred at 18° C. for 10 hours. The reaction mixture was concentratedunder reduced pressure to give a residue. The residue was purified bycolumn chromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1) to givecompound 65.5 (3 g, 7.02 mmol, 27% yield) as a white solid. LCMS (ESI)m/z: [M+H]+ calcd for C₂₂H₂₆N₃O₆: 428; found 428; RT=1.343 min.

To a solution of compound 65.5 (3 g, 7.02 mmol, 1 eq) in MeOH (20 mL)was added 10% palladium on carbon catalyst (100 mg) under N₂. Thesuspension was degassed under vacuum and purged with H₂ several times.The mixture was stirred under H₂ (50 psi) at 25° C. for hours. Thereaction mixture was filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Ethyl acetate) to give compound 65.4 (1.32 g, 4.47 mmol, 64% yield) as awhite solid. LCMS (ESI) m/z: [M+H]+ calcd for C₁₄H₂₂N₃O₄: 296; found296; RT=0.694 min. ¹H NMR (400 MHz, DMSO-d6) δ: 1.46 (s, 9H), 2.69-2.79(m, 1H), 2.81-2.91 (m, 1H), 3.54-3.62 (m, 4H), 6.85 (d, J=4.82 Hz, 1H),7.64 (s, 1H), 8.10 (d, J=4.82 Hz, 1H) and 9.71 (s, 1H) ppm.

To a mixture of 2-methoxy-2-methylpropanoic acid (633.59 mg, 5.36 mmol,1.2 eq) and EDCI (1.03 g, 5.36 mmol, 1.2 eq) in DMF (15 mL) was addedHOBt (724.7 mg, 5.36 mmol, 1.2 eq) in one portion at 18° C. under N₂.The mixture was stirred at 18° C. for 30 mins, and then was addedcompound 65.4 (1.32 g, 4.47 mmol, 1 eq) and DIPEA (1.73 g, 13.41 mmol,2.34 mL, 3 eq) in one portion, the mixture was stirred at 18° C. for 30mins. The reaction mixture was diluted with H₂O (10 mL) and extractedwith EtOAc (10 mL×3). The combined organic layers were washed with brine(10 mL), dried over Na₂SO₄, filtered and concentrated under reducedpressure. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1) to give compound 65.3 (1.5 g, 3.79mmol, 85% yield) as a white solid. LCMS (ESI) m/z: [M+H]+ calcd forC₁₉H₃₀N₃O₆: 396; found 396; RT=0.892 min. ¹H NMR (400 MHz, METHANOL-d4)δ: 1.31 (s, 3H), 1.37 (s, 3H), 1.56-1.64 (m, 9H), 3.16 (dd, J=13.89,9.26 Hz, 1H), 3.25 (s, 3H), 3.34 (dd, J=13.89, 4.85 Hz, 1H), 3.84 (s,3H), 4.82 (dd, J=9.15, 4.96 Hz, 1H), 6.96 (dd, J=5.07, 1.32 Hz, 1H),7.78-7.89 (m, 1H) and 8.18 (d, J=5.07 Hz, 1H) ppm.

To a solution of DIPA (281.48 mg, 2.78 mmol, 393.12 μL, 5.5 eq) in THF(10 mL) was added n-BuLi (2.5 M, 1.11 mL, 5.5 eq) at 0° C., the mixturewas stirred at 0° C. for 30 mins. To the mixture was added a solution ofcompound 65.3 (200 mg, 505.75 μmol, 1 eq) and chloroiodomethane (490.64mg, 2.78 mmol, 201.91 μL, 5.5 eq) in THF (10 mL) at −78° C. The mixturewas stirred at −78° C. for 30 mins. The reaction mixture was quenched byaddition aqueous saturated NH₄Cl (20 mL), and then extracted with EtOAc(20 mL×3). The combined organic layers were washed with aqueoussaturated Na₂SO₃ (30 mL), and brine (50 mL), dried over Na₂SO₄, filteredand concentrated under reduced pressure to give compound 65.2 (300 mg,crude) as yellow oil.

To a mixture of compound 65.2 (300 mg, 724.8 μmol, 1 eq) and2,3,5,6-tetrafluorophenol (120.37 mg, 724.8 μmol, 1 eq) in DMF (2 mL)was added DIEA (281.03 mg, 2.17 mmol, 378.74 μL, 3 eq) in one portion at18° C. under N₂. The mixture was stirred at 18° C. for 10 hours. Thereaction mixture was diluted with H₂O (4 mL) and extracted with EtOAc (4mL×3). The combined organic layers were washed with brine (5 mL), driedover Na₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1:1) to give compound 65.1 (20 mg, 36.8μmol, 5% yield) as a white solid. LCMS (ESI) m/z: [M+H]+ calcd forC₂₅H₃₀N₃O₆F₄: 544; found 544; RT=1.366 min.

To a solution of compound 65.1 (20 mg, 36.80 μmol, 1 eq) in DCM (5 mL)was added TFA (1 mL) in one portion at 18° C. under N₂. The mixture wasstirred at 18° C. for 10 mins. The reaction mixture was concentratedunder reduced pressure to give a residue. The residue was purified byprep-HPLC (basic condition) to give 65 (4 mg, 9 μmol, 25% yield) as awhite solid. LCMS (ESI) m/z: [M+H]+ calcd for C₂₀H₂₂N₃O₄F₄: 444; found444; RT=2.460 min. ¹H NMR (400 MHz, METHANOL-d4) δ: 1.02-1.35 (m, 6H),2.84 (br dd, J=13.89, 10.58 Hz, 1H), 3.06-3.26 (m, 4H), 4.61 (br d,J=9.04 Hz, 1H), 5.04-5.23 (m, 2H), 6.41-6.47 (m, 1H), 6.48-6.55 (m, 1H),7.07-7.21 (m, 1H) and 7.70-7.80 (m, 1H) ppm.

Example 66. Preparation ofN-(1-(6-aminopyridin-3-yl)-3-oxo-4-(2,3,6-trifluorophenoxy)butan-2-yl)cyclopentanecarboxamide(66)

To a solution of compound 66.9 (20 g, 167.9 mmol, 1 eq) in THF (250 mL)was added LiAlH₄ (12.74 g, 335.8 mmol, 2 eq) at 0° C. The mixture wasstirred at 0° C. for 1.5 hour. The reaction mixture was quenched byaddition of saturated sodium sulfate at 0° C. and added H₂O (200 mL)then extracted with ethyl acetate (200 mL×3). The combined organiclayers were washed with saturated brines (30 mL), dried over anhydroussodium sulfate, filtered and concentrated under reduced pressure to givecompound 66.8 (15 g, crude) as yellow oil. LCMS (ESI) m/z: [M+H]+ calcdfor C₆H₇N₂O: 123; found 123; RT=0.21 min.

To a solution of compound 66.8 (15 g, 122 mmol, 1 eq) in THF (100 mL)was added TEA (50 g, 591 mmol, 68 mL, 4 eq), Boc₂O (80.4 g, 368 mmol, 85mL, 3 eq) and DMAP (3 g, 24.5 mmol, 0.2 eq). The mixture was stirred at25° C. for 12 hours. The reaction mixture was quenched by addition H₂O200 mL at 25° C. and extracted with ethyl acetate (200 mL×3). Thecombined organic layers were washed with saturated brines (50 mL), driedover anhydrous sodium sulfate, filtered and concentrated under reducedpressure to give a crude product. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=3:1) to givecompound 66.7 (8 g, 24.82 mmol, 20% yield) as a white solid. LCMS (ESI)m/z: [M+H]+ calcd for C₁₆H₂₃N₂O₅: 323; found 323; RT=1.572 min.

To a solution of compound 66.7 (4 g, 12.4 mmol, 1 eq) in DCM (100 mL)was added DBU (3.8 g, 24.8 mmol, 3.8 mL, 2 eq) and compound 66.6 (4.1 g,12.4 mmol, 1 eq). The mixture was stirred at 25° C. for 0.5 hour. Thereaction mixture was quenched by addition of H₂O (100 mL) at 25° C. andextracted with ethyl acetate (100 mL×3). The combined organic layerswere washed with saturated brines (5 mL), dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give acrude product. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1) to give compound 66.5 (6 g, 11.37mmol, 92% yield) as yellow oil. LCMS (ESI) m/z: [M+H]+ calcd forC₂₇H₃₄N₃O₈: 528; found 528; RT=1.613 min.

To a solution of compound 66.5 (6 g, 11.37 mmol, 1 eq) in MeOH (400 mL)was added 10% palladium on carbon (1 g) under N₂. The suspension wasdegassed under vacuum and purged with H₂ several times. The mixture wasstirred under H₂ (50 psi) at 25° C. for 15 hours. The reaction mixturewas filtered, and the filter was concentrated to give compound 66.4 (3.3g, crude) as yellow oil. LCMS (ESI) m/z: [M+H]+ calcd for C₁₉H₃₀N₃O₆:396; found 396; RT=1.1445 min. ¹H NMR (400 MHz, chloroform-d) δ: 1.44(s, 25H), 2.83-2.97 (m, 1H), 3.07 (dd, 0.7=13.89, 5.51 Hz, 1H),3.68-3.78 (m, 4H), 7.18 (d, 0.7=8.16 Hz, 1H), 7.60 (dd, 0.7=8.16, 2.20Hz, 1H) and 8.32 (d, 0.7=1.98 Hz, 1H) ppm.

To a solution of compound 66.4 (2 g, 5.06 mmol, 1 eq) in DCM (30 mL) wasadded TEA (1.02 g, 10.12 mmol, 1.4 mL, 2 eq) and cyclopentane carbonylchloride (804.68 mg, 6.07 mmol, 738.24 μL, 1.20 eq). The mixture wasstirred at 25° C. for 5 min. The mixture was concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=1:1) to givecompound 66.3 (1.8 g, 3.66 mmol, 72% yield) as yellow oil. LCMS (ESI)m/z: [M+H]+ calcd for C₂₅H₃₈N₃O₇: 492; found 492; RT=1.552 min.

To a solution of DIPA (1.03 g, 10.17 mmol, 1.43 mL, 5 eq) in THF (10 mL)was added n-BuLi (2.5 M, 4.07 mL, 5 eq). The mixture was stirred at 0°C. for 0.5 hr under N₂. Then the mixture was added to the solution ofcompound 66.3 (1 g, 2.03 mmol, 1 eq) and chloroiodomethane (1.79 g,10.17 mmol, 738.3 μL, 5 eq) in THF (10 mL) was stirred at −78° C. for2.5 hours. The reaction mixture was quenched by addition saturated NH₄Cl(20 mL) at 25° C. and extracted with ethyl acetate (15 mL×3). Thecombined organic layers were washed with saturated Na₂SO₃ (10 mL) andsaturated NaHCO₃ (10 mL) and saturated brines (10 mL), dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to give compound 66.2 (1.6 g) as yellow oil.

To a solution of compound 66.2 (500 mg, 1.22 mmol, 1 eq) in DMF (5 mL)was added DIEA (472.93 mg, 3.66 mmol, 637.37 μL, 3 eq) and2,3,6-trifluorophenol (271 mg, 1.83 mmol, 1.5 eq). The mixture wasstirred at 25° C. for 15 hours. The reaction mixture was quenched byaddition saturated NaHCO₃ 20 mL at 25° C. and extracted with ethylacetate (20 mL×3). The combined organic layers were washed withsaturated brines (10 mL), dried over anhydrous sodium sulfate, filteredand concentrated under reduced pressure to give a crude product. Theresidue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=1:1) to give compound 66.1 (30 mg, 57.52 μmol, 5%yield) as yellow oil. LCMS (ESI) m/z: [M+H]+ calcd for C₂₆H₃₁N₃O₅F₃:522; found 522; RT=1.175 min.

To a solution of compound 66.1 (30 mg, 57.52 μmol, 1 eq) in DCM (5 mL)was added TFA (1 mL). The mixture was stirred at 25° C. for 15 hours.The reaction mixture was quenched by addition saturated NaHCO₃ (20 mL)at 25° C. and extracted with ethyl acetate (20 mL×3). The combinedorganic layers were washed with saturated brines (10 mL), dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to give a crude product. The residue was purified by prep-HPLC(TFA condition) to give 66 (4 mg, 9.5 μmol, 17% yield) was obtained as ayellow solid. LCMS (ESI) m/z: [M+H]+ calcd for C₂₁H₂₃N₃O₃F₃: 422; found422; RT=1.472 min. ¹H NMR (400 MHz, METHANOL-d₄) δ: 1.41-1.90 (m, 8H),2.63 (quin, J=7.66 Hz, 1H), 2.77 (dd, J=14.33, 9.70 Hz, 1H), 3.11-3.24(m, 1H), 4.86 (br s, 1H), 4.97-5.14 (m, 2H), 6.87-7.06 (m, 3H), 7.66 (s,1H) and 7.85 (dd, J=9.15, 1.87 Hz, 1H) ppm.

Example 67. Preparation ofN-(1-(6-aminopyridin-3-yl)-4-(2,6-difluorophenoxy)-3-oxobutan-2-yl)cyclopentanecarboxamide(67)

To a solution of compound 66.2 (500 mg, 1.22 mmol, 1 eq) in DMF (5 mL)was added DIEA (472.93 mg, 3.66 mmol, 637.37 μL, 3 eq) and2,6-difluorophenol (238.07 mg, 1.83 mmol, 1.5 eq). The mixture wasstirred at 25° C. for 15 hours. The reaction mixture was quenched byaddition of saturated NaHCO₃ (20 mL) at 25° C. and extracted with ethylacetate (20 mL×3). The combined organic layers were washed withsaturated brines (10 mL), dried over anhydrous sodium sulfate, filteredand concentrated under reduced pressure to give a crude product. Theresidue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=1:1) to give compound 67.1 (30 mg, 59.6 μmol, 5%yield) as yellow oil. LCMS (ESI) m/z: [M+H]+ calcd for C₂₆H₃₂N₃O₅F₂:504; found 504; RT=1.112 min.

To a solution of compound 67.1 (30 mg, 59.58 μmol, 1 eq) in DCM (5 mL)was added TFA (1 mL). The mixture was stirred at 25° C. for 15 hours.The reaction mixture was quenched by addition saturated NaHCO₃ (20 mL)at 25° C. and extracted with ethyl acetate (20 mL×3). The combinedorganic layers were washed with saturated brines (10 mL), dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to give a crude product. The residue was purified by prep-HPLC(TFA condition) to give 67 (4 mg, 9.92 μmol, 17% yield) as a yellowsolid. LCMS (ESI) m/z: [M+H]+ calcd for C₂₁H₂₄N₃O₃F₂: 404; found 404;RT=1.422 min. ¹H NMR (400 MHz, METHANOL-d₄) δ: 1.42-1.94 (m, 8H),2.52-2.69 (m, 1H), 2.76 (dd, 0.7=14.44, 9.81 Hz, 1H), 3.09-3.25 (m, 1H),4.90-5.04 (m, 3H), 6.87-7.13 (m, 4H), 7.65 (s, 1H) and 7.85 (dd, J=9.26,1.98 Hz, 1H) ppm.

Example 68. Preparation ofN-(1-(6-aminopyridin-3-yl)-4-(isoxazol-3-yloxy)-3-oxobutan-2-yl)cyclopentanecarboxamide(68)

To a solution of compound 66.2 (400 mg, 975.83 μmol, 1 eq) in DMF (5 mL)was added K₂CO₃ (404.6 mg, 2.93 mmol, 3 eq) and isoxazol-3-ol (124.51mg, 1.46 mmol, 1.5 eq). The mixture was stirred at 25° C. for 15 hours.The reaction mixture was quenched by addition saturated NaHCO₃ 20 mL at25° C. and extracted with ethyl acetate (20 mL×3). The combined organiclayers were washed with saturated brines (10 mL), dried over anhydroussodium sulfate, filtered and concentrated under reduced pressure to givea crude product. The residue was purified by column chromatography(SiO₂, Petroleum ether/Ethyl acetate=1:1) to give compound 68.1 (50 mg)as yellow oil.

A mixture of compound 68.1 (100 mg, 65.43 μmol, 1 eq) in TFA (1 mL) andDCM (5 mL) was stirred at 25° C. for 12 hours. The mixture wasconcentrated in vacuum. The residue was purified by preparativescale-HPLC (TFA condition) to give 68 (2 mg, 5.58 μmol, 9% yield) as awhite solid. LCMS (ESI) m/z: [M+H]+ calcd for C₁₈H₂₃N₄O₄: 359; found359; RT=1.169 min. ¹H NMR (400 MHz, METHANOL-d₄) δ: 1.43-1.91 (m, 10H),2.56-2.72 (m, 1H), 2.79 (dd, 0.7=14.44, 9.37 Hz, 1H), 3.09-3.23 (m, 1H),4.81 (dd, J=9.37, 5.40 Hz, 1H), 5.05 (d, J=2.43 Hz, 2H), 6.17 (d,0.7=1.76 Hz, 1H), 6.93 (d, J=9.04 Hz, 1H), 7.67 (s, 1H), 7.84 (dd,0.7=9.15, 1.87 Hz, 1H) and 8.39 (d, 0.7=1.76 Hz, 1H) ppm.

Example 69. Preparation ofN-(1-(2-aminopyridin-4-yl)-3-oxo-4-(2,3,6-trifluorophenoxy)butan-2-yl)cyclopentanecarboxamide(69)

To a solution of compound 69.11 (50 g, 362 mmol, 1 eq) in MeOH (600 mL)was added SOCl₂ (172.27 g, 1.45 mol, 105.04 mL, 4 eq) dropwise at 0° C.under N₂, then heated to 18° C. and stirred for 10 hours. The reactionmixture was concentrated under reduced

pressure to give compound 69.10 (60 g, crude) as a white solid. LCMS(ESI) m/z: [M+H]+ calcd for C₇H₈N₂O₂: 152; found 153; RT=0.214 min.

To a mixture of compound 69.10 (60 g, 394.35 mmol, 1 eq) and DMAP (2.41g, 19.72 mmol, 0.05 eq) in t-BuOH (600 mL) and ACETONE (200 mL) wasadded Boc₂O (344.26 g, 1.58 mol, 362.38 mL, 4 eq) dropwise at 18° C.under N₂. The mixture was stirred at 18° C. for 15 hours. The solutionwas diluted with pentane (200 mL), cooled in the refrigerator for 3hours and filtered to obtain compound 69.9 (42 g, 166.5 mmol, 42% yield)as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ: 1.38-1.54 (m, 9H),3.82-3.96 (m, 3H), 7.44 (dd, J=5.07, 1.41 Hz, 1H), 8.32 (s, 1H), 8.42(d, J=5.14 Hz, 1H) and 10.11 (s, 1H) ppm.

To a solution of compound 69.9 (42 g, 166.5 mmol, 1 eq) in THF (800 mL)was added LiAlH₄ (12.64 g, 333 mmol, 2 eq) portion-wise at 0° C. underN₂. The mixture was stirred at 0° C. for 1 hour, then heated to 18° C.and stirred at 18° C. for 14 hours. The reaction mixture was quenched byaddition of 8% NaOH (15 mL), filtered and then diluted with H₂O (1000mL) and extracted with EtOAc (500 mL×3). The combined organic layerswere washed with brine (1000 mL), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=2:1) to give compound 69.8 (15 g, 67 mmol, 40% yield) as a whitesolid. ¹H NMR (400 MHz, DMSO-d6) δ: 1.47 (s, 9H), 4.50 (d, J=5.70 Hz,2H), 5.40 (t, J=5.92 Hz, 1H), 6.89-6.99 (m, 1H), 7.81 (s, 1H), 8.14 (d,J=5.70 Hz, 1H) and 9.71 (s, 1H) ppm.

To a solution of compound 69.8 (15 g, 67 mmol, 1 eq) in DCM (150 mL) wasadded Dess-Martin periodinane (42.55 g, 100.33 mmol, 31.06 mL, 1.5 eq)portion-wise at 18° C. under N₂. The mixture was stirred at 18° C. for 2hours. The reaction mixture was diluted with H₂O (60 mL) and extractedwith DCM (50 mL×3). The combined organic layers were washed with brine(100 mL), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=5:1) to givecompound 69.7 (9 g, 40.5 mmol, 60% yield) as a white solid. ¹H NMR (400MHz, chloroform-d) δ ppm 1.55-1.62 (m, 9H), 7.40 (dd, J=5.26, 1.32 Hz,1H), 8.44-8.57 (m, 2H), 9.38 (s, 1H) and 10.01-10.11 (m, 1H) ppm.

To a mixture of compound 69.7 (5 g, 22.5 mmol, 1 eq) and compound 69.6(7.45 g, 22.5 mmol, 1 eq) in DCM (60 mL) was added2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (DBU) (6.85 g, 45 mmol,6.78 mL, 2 eq) in one portion at 18° C. under N₂. The mixture wasstirred at 18° C. for 10 hours. The reaction mixture was concentratedunder reduced pressure to give a residue. The residue was purified bycolumn chromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1) to givecompound 69.5 (5.7 g, 13.33 mmol, 59% yield) as a white solid.

To a solution of compound 69.5 (5.7 g, 13.33 mmol, 1 eq) in MeOH (20 mL)was added 10% palladium on carbon catalyst (100 mg) under N₂. Thesuspension was degassed under vacuum and purged with H₂ several times.The mixture was stirred under H₂ (50 psi) at 25° C. for 10 hours. Thereaction mixture was filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Ethyl acetate) to give compound 69.4 (2.8 g, 9.48 mmol, 71% yield) as awhite solid.

To a mixture of cyclopentanecarboxylic acid (680.21 mg, 5.96 mmol,647.82 μL, 1 eq) and EDCI (1.26 g, 6.56 mmol, 1.1 eq) in DMF (20 mL) wasadded HOBt (885.75 mg, 6.56 mmol, 1.1 eq) in one portion at 0° C. underN₂. The mixture was stirred at 0° C. for 1 hour, then the mixture wasadded dropwise a solution of compound 69.4 (1.76 g, 5.96 mmol, 1 eq) inDMF (5 mL), then the mixture was added dropwise DIPEA (2.31 g, 17.88mmol, 3.12 mL, 3 eq) and stirred at 0° C. for 1 hour. The reactionmixture was diluted with H₂O (10 mL) and extracted with EtOAc (10 mL×3).The combined organic layers were washed with brine (15 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=3:1) to give compound 69.3 (1.8 g, 4.6mmol, 77% yield) as a white solid.

To a solution of DIPA (1.42 g, 14.05 mmol, 1.99 mL, 5.5 eq) in THF (40mL) was added n-BuLi (2.5 M, 5.62 mL, 5.5 eq) at 0° C., the mixture wasstirred at 0° C. for 30 mins. The mixture was added a solution ofcompound 69.3 (1 g, 2.55 mmol, 1 eq) and chloroiodomethane (2.48 g,14.05 mmol, 1.02 mL, 5.5 eq) in THF (40 mL) at −78° C. The mixture wasstirred at −78° C. for 30 mins. The reaction mixture was quenched byaddition aqueous saturated NH₄Cl (40 mL) and then extracted with EtOAc(50 mL×3). The combined organic layers were washed with aqueoussaturated Na₂SO₃ (80 mL), and brine (50 mL), dried over Na₂SO₄, filteredand concentrated under reduced pressure to give compound 69.2 (1.4 g) asyellow oil.

To a mixture of compound 69.2 (300 mg, 731.87 μmol, 1 eq) and2,3,6-trifluorophenol (108.38 mg, 731.87 μmol, 1 eq) in DMF (2 mL) wasadded DIEA (283.76 mg, 2.2 mmol, 382.43 μL, 3 eq) in one portion at 18°C. under N₂. The mixture was stirred at 18° C. for 10 hours. The mixturewas purified by semi-preparative HPLC (TFA condition) to give compound69.1 (80 mg, 153.4 μmol, 21% yield) as a white solid. LCMS (ESI) m/z:[M+H]+ calcd for C₂₆H₃₀N₃O₅F₃: 522; found 522; RT=1.366 min.

To a solution of compound 69.1 (80 mg, 153.4 μmol, 1 eq) in DCM (5 mL)was added TFA (1 mL) in one portion at 18° C. under N₂. The mixture wasstirred at 18° C. for 1 hour. The reaction mixture was concentratedunder reduced pressure to give a residue. The residue was purified bysemi-preparative HPLC (TFA condition) to give 69 (40 mg, 95 μmol, 62%yield) as a white solid. LCMS (ESI) m/z: [M+H]+ calcd for C₂₁H₂₃N₃O₃F₃:442; found 442; RT=1.581 min. ¹H NMR (400 MHz, DMSO-d6) δ: 1.34-1.49 (m,3H), 1.49-1.59 (m, 3H), 1.60-1.73 (m, 2H), 2.51 (br s, 1H), 2.75 (dd,J=13.67, 10.58 Hz, 1H), 3.10 (br dd, J=13.78, 4.08 Hz, 1H), 4.61-4.74(m, 1H), 5.11 (d, J=1.54 Hz, 2H), 6.70 (s, 1H), 6.74 (d, J=6.61 Hz, 1H),7.08-7.26 (m, 2H), 7.84 (d, J=6.62 Hz, 1H), 7.96 (br s, 2H) and 8.32 (d,J=8.16 Hz, 1H) ppm.

Example 70. Preparation ofN-(1-(2-aminopyridin-4-yl)-4-(isoxazol-3-yloxy)-3-oxobutan-2-yl)cyclopentanecarboxamide(70)

To a mixture of compound 69.2 (300 mg, 732 μmol, 1 eq) and isoxazol-3-ol(62.25 mg, 732 μmol, 1 eq) in DMF (2 mL) was added DIEA (378.35 mg, 2.93mmol, 510 μL, 4 eq) in one portion at 18° C. under N₂. The mixture wasstirred at 18° C. for 10 hours. The mixture was purified bysemi-preparative scale HPLC (TFA condition) to give compound 70.1 (50mg, 109 μmol, 15% yield) as a white solid. LCMS (ESI) m/z: [M+H]+ calcdfor C₂₃H₃₁N₄O₆: 459; found 459; RT=1.233 min.

To a solution of compound 70.1 (50 mg, 109 μmol, 1 eq) in DCM (5 mL) wasadded TFA (1 mL) in one portion at 18° C. under N₂. The mixture wasstirred at 18° C. for 1 hour. The reaction mixture was concentratedunder reduced pressure to give a residue. The residue was purified bysemi-preparative scale HPLC (TFA condition) to give 70 (15 mg, 41.85μmol, 38% yield) as a white solid. LCMS (ESI) m/z: [M+H]+ calcd forC₁₈H₂₃N₄O₄: 359; found 359; RT=1.765 min. ¹H NMR (400 MHz, DMSO-d6) δ:1.39-1.51 (m, 2H), 1.52-1.62 (m, 2H), 1.63-1.77 (m, 2H), 2.53-2.59 (m,1H), 2.78 (br dd, J=13.56, 10.47 Hz, 1H), 3.15 (br dd, J=13.89, 3.97 Hz,1H), 4.66-4.76 (m, 1H), 5.09 (s, 1H), 6.38 (d, J=1.76 Hz, 1H), 6.73 (s,1H), 6.78 (d, J=6.61 Hz, 1H), 7.85 (d, J=6.61 Hz, 1H), 7.89 (br s, 1H),8.36 (br d, J=8.38 Hz, 1H) and 8.68 (d, J=1.76 Hz, 1H) ppm.

Example 71. Preparation ofN-(5-(2-aminopyridin-4-yl)-1-(isoxazol-3-yloxy)-2-oxopentan-3-yl)cyclopentanecarboxamide(71)

To a mixture of compound 71.8 (5 g, 28.9 mmol, 1 eq) and Boc₂O (25.23 g,115.6 mmol, 26.6 mL, 4 eq) in DMF (50 mL) was added TEA (14.62 g, 144.5mmol, 20.1 mL, 5 eq) and DMAP (353.1 mg, 2.89 mmol, 0.1 eq) in oneportion at 25° C. under N₂. The mixture was stirred at 25° C. for 10hours. The reaction mixture was diluted with H₂O (50 mL) and extractedwith EtOAc (50 mL×3). The combined organic layers were washed with brine(100 mL), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=3:1) to givecompound 71.7 (2.7 g, 7.23 mmol, 25% yield) as a white solid. ¹H NMR(400 MHz, DMSO-d6) δ: 7.41 (d, J=8.93 Hz, 1H), 8.12 (dd, J=8.56, 2.45Hz, 1H), 8.59 (d, J=2.20 Hz, 1H) and 1.39 (s, 17H) ppm.

To a solution of compound 71.6 in dioxane (20 mL) was added 9-BBN (0.5M, 15.9 mL, 2.2 eq) in one portion at 80° C. under N₂. The mixture wasstirred at 80° C. for 2 hours, then to the mixture was added Pd(dppf)Cl₂(264.20 mg, 361.07 μmol, 0.1 eq), CsF (1.65 g, 10.83 mmol, 399.36 μL, 3eq) and compound 71.7 (1.62 g, 4.33 mmol, 1.2 eq). This was stirred at80° C. for 8 hours. The reaction mixture was diluted with H₂O (20 mL)and extracted with EtOAc (20 mL×3). The combined organic layers werewashed with brine (20 mL), dried over Na₂SO₄, filtered and concentratedunder reduced pressure to give a residue. The residue was purified bycolumn chromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1) to givecompound 71.5 (1 g, 1.84 mmol, 51% yield) as a white solid. LCMS (ESI)m/z: [M+H]+ calcd for C₂₈H₃₈N₃O₈: 544; found 544; RT=1.615 min.

To a solution of compound 71.5 (1 g, 1.84 mmol, 1 eq) in MeOH (50 mL)was added 10% Pd on carbon catalyst (100 mg) under N₂. The suspensionwas degassed under vacuum and purged with H₂ several times. The mixturewas stirred under H₂ atmosphere (50 psi) at 25° C. for hours. Thereaction mixture was filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Ethyl acetate) to give compound 71.4 (400 mg, 977 μmol, 53% yield) as awhite solid. ¹H NMR (400 MHz, METHANOL-d4) δ: 0.99 (s, 18H), 1.44-1.56(m, 1H), 1.58-1.70 (m, 1H), 2.38 (t, J=7.95 Hz, 2H), 2.87-2.94 (m, 2H),2.89-2.92 (m, 1H), 3.03 (dd, J=7.21, 5.75 Hz, 1H), 6.86 (d, J=8.19 Hz,1H), 7.40 (dd, J=8.19, 2.45 Hz, 1H) and 7.92 (d, J=2.08 Hz, 1H) ppm.

To a mixture of cyclopentanecarboxylic acid (111.50 mg, 0.977 μmol,106.2 μL, 1 eq) and EDCI (224.72 mg, 1.17 mmol, 1.2 eq) in DMF (10 mL)was added HOBt (158.39 mg, 1.17 mmol, 1.2 eq) in one portion at 18° C.under N₂. The mixture was stirred at 25° C. for 30 mins, then compound71.4 (400 mg, 976.86 μmol, 1 eq) and DIPEA (378.76 mg, 2.93 mmol, 510.45μL, 3 eq) were added in one portion. This mixture was stirred at 25° C.for 30 mins. The reaction was diluted with H₂O (10 mL) and extractedwith EtOAc (10 mL×3). The combined organic layers were washed with brine(10 mL), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1) to givecompound 71.3 (600 mg) as a white solid. LCMS (ESI) m/z: [M+H]+ calcdfor C₂₆H₄₀O₇N₃: 506; found 506; RT=1.316 min.

To a solution of DIPA (600.41 mg, 5.93 mmol, 838.6 μL, 5 eq) in THF (10mL) was added n-BuLi (2.5 M, 2.37 mL, 5 eq) at 0° C.; the mixture wasstirred at 0° C. for 30 mins. The mixture was added a solution ofchloroiodomethane (1.05 g, 5.93 mmol, 430.69 μL, 5 eq) and compound 71.3(600 mg, 1.19 mmol, 1 eq) in THF (10 mL) at −78° C. This was stirred at−78° C. for 30 mins. The reaction mixture was quenched by addition ofsaturated aqueous NH₄Cl (20 mL) and then extracted with EtOAc (20 mL×3).The combined organic layers were washed with saturated aqueous Na₂SO₃(30 mL), and brine (50 mL), dried over Na₂SO₄, filtered and concentratedunder reduced pressure. The residue was purified by semi-preparativescale HPLC (TFA condition) to give compound 71.2 (100 mg, 190.8 μmol,16% yield) as a white solid.

To a mixture of compound 71.2 (70 mg, 133.6 μmol, 1 eq) andisoxazol-3-ol (11.36 mg, 133.6 μmol, 1 eq) in DMF (2 mL) was added DIEA(17.26 mg, 133.6 μmol, 23.3 μL, 1 eq) in one portion at 25° C. under N₂.The mixture was stirred at 25° C. for 10 hours. The reaction mixture wasdiluted with H₂O (10 mL) and extracted with EtOAc (10 mL×3). Thecombined organic layers were washed with brine (20 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure to givecompound 71.1 (80 mg) as a yellow oil.

To a solution of compound 71.1 (79.73 mg, 139.2 μmol, 1 eq) in DCM (5mL) was added TFA (139.2 μmol, 10.31 μL, 1 eq) in one portion at 25° C.under N₂. The mixture was stirred at 25° C. for 1 hour. The reactionmixture was concentrated under reduced pressure; the residue waspurified by semi-preparative scale HPLC (neutral condition) to give 71(2 mg, 5.13 μmol, 4% yield, 95.5% purity) as a white solid. LCMS (ESI)m/z: [M+H]+ calcd for C₁₉H₂₄N₄O₄: 373; found 373; RT=5.70 min. ¹H NMR(400 MHz, METHANOL-d4) δ: 1.72 (m, 2H), 1.74 (m, 4H), 1.87 (m, 3H), 2.10(m, 1H), 2.53 (m, 2H), 2.61 (m, 1H), 4.46 (dd, J=9.60, 4.40 Hz, 1H),4.97-5.07 (m, 1H), 6.13 (s, 1H), 6.57 (d, J=8.40 Hz, 1H), 7.40 (dd,J=8.40, 2.45 Hz, 1H), 7.72 (s, 1H) and 8.36 (dd, J=5.20, 1.60 Hz, 1H)ppm.

Example 72. Preparation ofN-(1-(6-aminopyridin-3-yl)-4-((1,1,1,3,3,3-hexafluoropropan-2-yl)oxy)-3-oxobutan-2-yl)-2-methoxy-2-methylpropanamide(72)

To a solution of compound 72.9 (10 g, 83.9 mmol, 1 eq) in THF (250 mL)was added LAH (6.37 g, 167.9 mmol, 2 eq) at 0° C. The mixture wasstirred at 0° C. for 1.5 hour. The reaction mixture was quenched byaddition saturated sodium sulfate at 0° C. and H₂O (200 mL) was added;then this was extracted with ethyl acetate (200 mL×3). The combinedorganic layers were washed with saturated brine (30 mL), dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to give the product compound 72.8 (6 g, crude) as a yellowsolid. LCMS (ESI): m/z: [M+H]+ calcd for C₆H₇N₂O: 123; found 123;RT=0.276 min.

To a solution of compound 72.8 (6 g, 49.14 mmol, 1 eq) in THF (100 mL)was added TEA (19.89 g, 196.53 mmol, 4 eq), Boc₂O (32.16 g, 147.39 mmol,3 eq) and DMAP

(1.2 g, 9.9 mmol, 0.2 eq). The mixture was stirred at 25° C. for 12hours. The reaction mixture was quenched by addition of H₂O (200 mL) at25° C. and extracted with ethyl acetate (200 mL×3). The combined organiclayers were washed with saturated brines (50 mL), dried over anhydroussodium sulfate, filtered and concentrated under reduced pressure to givea crude product. The residue was purified by column chromatography(SiO₂, Petroleum ether/Ethyl acetate=3:1) to give compound 72.7 (5 g,15.51 mmol, 32% yield) as a yellow solid. LCMS (ESI) m/z: [M+H]+ calcdfor C₁₆H₂₃N₂O₅: 323; found 323; RT=1.532 min. ¹H NMR (400 MHz,chloroform-d) δ: 1.52 (br d, J=3.97 Hz, 28H), 7.59-7.74 (m, 1H),8.10-8.28 (m, 1H), 8.87 (br s, 1H), 10.07 (br d, J=3.97 Hz, 1H) ppm.

To a solution of compound 72.7 (5 g, 15.5 mmol, 1 eq) in DCM (60 mL) wasadded DBU (4.7 g, 30.4 mmol, 2 eq) and compound 72.6 (5.1 g, 15.5 mmol,1 eq). The mixture was stirred at 25° C. for 2 hours. The reactionmixture was quenched by addition of H₂O (100 mL) at 25° C. and extractedwith ethyl acetate (100 mL×3). The combined organic layers were washedwith brine (5 mL), dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure to give a crude product. The residuewas purified by column chromatography (SiO2, Petroleum ether/Ethylacetate=2:1) to give compound 72.5 (3 g, 5.7 mmol, 37% yield) as yellowoil. LCMS (ESI) m/z: [M+H]+ calcd for C₂₇H₃₄N₃O₈: 528; found 528;RT=1.604 min.

To a solution of compound 72.5 (3 g, 5.69 mmol, 1 eq) in MeOH (400 mL)was added 10% Pd on carbon catalyst (1 g) under N₂. The suspension wasdegassed under vacuum and purged with H₂ several times. The mixture wasstirred under H₂ (50 psi) at 25° C. for 15 hours. The reaction mixturewas filtered, and the filtrate was concentrated, compound 76.4 (1.5 g)was obtained as yellow oil. LCMS (ESI) m/z: [M+H]+ calcd for C₁₉H₃₀N₃O₆:396; found 396; RT=1.084 min.

To a solution of 2-methoxy-2-methylpropanoic acid (448.09 mg, 3.79 mmol,1 eq) in DMF (20 mL) was added HOBt (563.78 mg, 4.17 mmol, 1.1 eq) andEDCI (799.86 mg, 4.17 mmol, 1.1 eq). The mixture was stirred at 25° C.for 1 hr. To the mixture was added compound 72.4 (1.5 g, 3.79 mmol, 1eq) and DIPEA (1.96 g, 15.17 mmol, 2.64 mL, 4 eq). The mixture wasstirred at 25° C. for 14 hrs. The reaction mixture was quenched byaddition of H₂O (30 mL) and was extracted with ethyl acetate (30 mL×3).The combined organic layers were washed with brine (5 mL), dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1) to give compound 72.3 (1.25 g, 2.52mmol, 67% yield) as yellow oil. LCMS (ESI) m/z: [M+H]+ calcd forC₂₄H₃₈N₃O₈: 496; found 496; RT=1.260 min.

To a solution of DIPA (1.40 g, 13.87 mmol, 1.96 mL, 5.5 eq) in THF (10mL) was added n-BuLi (2.5 M, 5.55 mL, 5.5 eq). The mixture was stirredat 0° C. for 0.5 hr under N₂. The mixture was added to compound 72.3(1.25 g, 2.52 mmol, 1 eq) and chloroiodomethane (2.45 g, 13.87 mmol,1.01 mL, 5.5 eq) in THF (10 mL) was stirred at −78° C. for 2.5 hrs. Thereaction mixture was quenched by addition of saturated NH₄Cl (20 ml) at25° C. and extracted with ethyl acetate (15 mL×3). The combined organiclayers were washed with saturated Na₂SO₃ (10 mL) and saturated NaHCO₃(10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, filteredand concentrated under reduced pressure. The residue was purified bycolumn chromatography (SiO₂, Petroleum ether/Ethyl acetate=0:1) to givecompound 72.2 (100 mg, 194.55 μmol, 8% yield) as yellow oil. LCMS (ESI)m/z: [M+H]+ calcd for C₂₄H₃₇N₃O₇Cl: 514; found 514; RT=0.971 min.

To a solution of compound 72.2 (40 mg, 77.82 μmol, 1 eq) in1,1,1,3,3,3-hexafluoropropan-2-ol (800 mg, 4.76 mmol, 100 μL, 61.18 eq)was added DIEA (60.35 mg, 466.92 μmol, 81.33 μL, 6 eq). The mixture wasstirred at 25° C. for 12 hrs. The reaction mixture was purified bycolumn chromatography (SiO₂, Petroleum ether/Ethyl acetate=1:1) to givecompound 72.1 (20 mg, 31 μmol, 40% yield) as yellow oil. LCMS (ESI) m/z:[M+H]+ calcd for C₂₂H₃₀N₃O₆F₆: 646; found 546; RT=1.301 min.

Compound 72.1 was added into DCM (5 mL) and TFA (1 mL), the mixture wasstirred at 25° C. for 1 hour. The mixture was concentrated under reducedpressure to give a residue. The residue was purified by semi-preparativescale HPLC (TFA condition) to give 72 (2 mg, 3.58 μmol, 12% yield, TFA)as a white solid. LCMS (ESI) m/z: [M+H]+ calcd for C₁₇H₂₂N₃O₇F₆: 446;found 446; RT=0.925 min. ¹H NMR (400 MHz, METHANOL-d₄) δ: 1.34 (d,J=9.54 Hz, 6H), 2.23 (s, 3H), 3.32-3.34 (m, 3H), 4.91-4.95 (m, 3H), 5.17(dq, J=11.95, 6.04 Hz, 1H), 6.92 (d, J=9.17 Hz, 1H), 7.63 (s, 1H) and7.69 (dd, J=9.23, 2.02 Hz, 1H) ppm.

Example 73. Preparation ofN-(1-(2-aminopyridin-4-yl)-4-((1,1,1,3,3,3-hexafluoropropan-2-yl)oxy)-3-oxobutan-2-yl)-2-methoxy-2-methylpropanamide(73)

To a solution of compound 73.11 (50 g, 362 mmol, 1 eq) in MeOH (600 mL)was added SOCl₂ (172.27 g, 1.45 mol, 105.04 mL, 4 eq) drop wise at 0° C.under N₂, then stirred at 18° C. for 10 hours. The reaction mixture wasconcentrated under reduced pressure to give compound 73.10 (60 g, crude)as a white solid. LCMS (ESI) m/z: [M+H]+ calcd for C₇H₉N₂O₂: 153; found153; RT=0.214 min.

To a mixture of compound 73.10 (60 g, 394.35 mmol, 1 eq) and DMAP (2.41g, 19.72 mmol, 0.05 eq) in t-BuOH (600 mL) and ACETONE (200 mL) wasadded Boc₂O (344.26 g, 1.58 mol, 362.38 mL, 4 eq) dropwise. The mixturewas stirred for 15 hours. The solution was diluted with pentane (200ml), cooled in the refrigerator for 3 hours then filtered to givecompound 73.9 (42 g, 166.49 mmol, 42% yield) as a white solid. LCMS(ESI) m/z: [M+H]+ calcd for C₁₂H₁₇N₂O₄: 253; found 253; RT=1.562 min.

To a solution of compound 73.9 (42 g, 166.49 mmol, 1 eq) in THF (800 mL)was added LiAlH₄ (12.64 g, 332.98 mmol, 2 eq) portion wise at 0° C.under N₂. The mixture was

stirred at 0° C. for 1 hours, then stirred for 14 hours. The reactionmixture was quenched by addition 8% NaOH (15 ml), filtered and thendiluted with H₂O (1000 mL) and extracted with EtOAc (500 mL×3). Thecombined organic layers were washed with brine (1000 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=2:1) to give compound 73.8 (15 g, 66.89mmol, 40% yield) as a white solid. LCMS (ESI) m/z: [M+H]+ calcd forC₁₁H₁₇N₂O₃: 225; found 225; RT=0.833 min.

To a solution of compound 73.8 (15 g, 66.89 mmol, 1 eq) in DCM (150 mL)was added Dess-Martin periodinane (42.55 g, 100.33 mmol, 31.06 mL, 1.5eq) portion-wise at 18° C. under N₂. The mixture was stirred for 2hours. The reaction mixture was diluted with H₂O (60 mL) and extractedwith DCM 150 mL (50 mL×3). The combined organic layers were washed withbrine (100 mL) (100 mL), dried over Na₂SO₄, filtered and concentratedunder reduced pressure to give a residue. The residue was purified bycolumn chromatography (SiO₂, Petroleum ether/Ethyl acetate=5:1) to givecompound 73.7 (9 g, 40.5 mmol, 61% yield) as a white solid.

To a mixture of compound 5 (2 g, 9 mmol, 1 eq) and compound 73.6 (2.98g, 9 mmol, 1 eq) in DCM (20 mL) was added DBU (2.74 g, 18 mmol, 2.71 mL,2 eq) in one portion. The mixture was stirred at 18° C. for 10 hours.The reaction mixture was filtered and concentrated under reducedpressure to give a residue. The residue was purified by MPLC (SiO₂,Petroleum ether/Ethyl acetate=10/l to 0:1) to give compound 73.5 (3 g,7.02 mmol, 78% yield) as a white solid. ¹H NMR (400 MHz, chloroform-d)δ: 8.41-8.47 (m, 1H), 8.39-8.49 (m, 1H), 8.26-8.33 (m, 1H), 8.25-8.33(m, 1H), 8.10 (br d, J=14.77 Hz, 1H), 8.06-8.14 (m, 1H), 7.32-7.50 (m,5H), 7.04-7.10 (m, 1H), 7 (d, J=5.29 Hz, 1H), 6.97-7.10 (m, 1H), 6.85(br d, 0.7=4.41 Hz, 1H), 6.76-6.91 (m, 1H), 6.38 (s, 1H), 5.17 (s, 1H),3.91 (s, 2H), 3.95 (s, 1H), 1.59 (s, 5H) and 1.61 (s, 3H) ppm.

To a solution of compound 73.5 (3 g, 7.02 mmol, 1 eq) in MeOH (300 mL)was added 10% palladium on carbon catalyst Pd/C (300 mg) under N₂. Thesuspension was degassed under vacuum and purged with H₂ several times.The mixture was shaken under H₂ (50 psi) at 25° C. for 20 hours. Thereaction mixture was filtered. The filtrate was concentrated underreduced pressure to give a residue. The residue was purified by MPLC(SiO₂, Petroleum ether/Ethyl acetate=10/1 to 1:1) to give compound 73.4(1.4 g, 4.74 mmol, 68% yield) as a white solid. LCMS (ESI) m/z: [M+H]+calcd for C₁₄H₂₂N₃O₄: 296; found 296; RT=0.654 min.

To a mixture of 2-methoxy-2-methylpropanoic acid (560 mg, 4.74 mmol, 1eq) and HOBt (704.6 mg, 5.21 mmol, 1.1 eq) in DMF (20 mL) was added EDCI(999.6 mg, 5.21 mmol, 1.1 eq) in one portion at 25° C. under N₂. Themixture was stirred at 25° C. for 1 hour, then to the mixture was addedcompound 73.4 (1.4 g, 4.74 mmol, 1 eq) and DIPEA (1.84 g, 14.22 mmol,2.48 mL, 3 eq) in one portion, and stirred at 25° C. for 9 hours. Thereaction mixture was quenched by addition of H₂O (20 mL) and thenextracted with EtOAc 60 mL (20 mL×3). The combined organic layers werewashed with H₂O (20 mL×3) and brine (20 mL) then dried over Na₂SO₄,filtered and concentrated. The residue was purified by MPLC (SiO₂,Petroleum ether/Ethyl acetate=10/l to 0:1) to give compound 73.3 (1.2 g,3.03 mmol, 64% yield) as a white solid. ¹H NMR (400 MHz, chloroform-d)δ: 8.57 (s, 1H), 8.18 (d, J=5.14 Hz, 1H), 7.82 (s, 1H), 7.09 (br d,J=8.19 Hz, 1H), 6.74 (dd, J=5.26, 1.47 Hz, 1H), 4.83-4.90 (m, 1H), 3.77(s, 3H), 3.20 (s, 3H), 3.17 (d, J=5.38 Hz, 1H), 3.01-3.09 (m, 1H), 1.52(s, 9H), 1.34 (s, 3H) and 1.31 (s, 3H) ppm.

To a solution of DIPA (633.32 mg, 6.26 mmol, 884.52 μL, 5.5 eq) in THF(20 mL) was added n-BuLi (2.5 M, 2.5 mL, 5.5 eq) at 0° C., the mixturewas stirred at 0° C. for 30 mins. To the mixture was added a solution ofcompound 73.3 (0.45 g, 1.14 mmol, 1 eq) and chloroiodomethane (1.10 g,6.26 mmol, 454.29 μL, 5.5 eq) in THF (20 mL) at −78° C. The mixture wasstirred at −78° C. for 1.5 h. The reaction mixture was quenched byaddition of NH₄Cl (20 mL) and extracted with EtOAc (20 mL×3), Thecombined organic layers were washed with saturated brines (20 mL), driedover Na₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1:1) to give compound 73.2 (100 mg, 241.61μmol, 21% yield) as a white solid.

To a solution of compound 73.2 (40 mg, 96.64 μmol, 1 eq) in1,1,1,3,3,3-hexafluoropropan-2-ol (64.96 mg, 386.57 μmol, 100 μL, 4 eq)was added DIPEA (0.5 M, 193.29 μL, 1 eq) in one portion at 25° C. underN₂. The mixture was stirred at 25° C. for 20 min. The residue waspurified by semi-preparative scale HPLC (TFA condition) to give compound73.1 (5 mg, 9.17 μmol, 10% yield) as a white solid.

To a solution of compound 73.1 (5 mg, 9.17 μmol, 1 eq) in DCM (2 mL) wasadded TFA (0.5 mL) in one portion at 25° C. under N₂. The mixture wasstirred at 25° C. for 1 min. Then the reaction mixture was concentratedunder reduced pressure to give 73 (2 mg, 4.49 μmol, 50% yield) as awhite solid. LCMS (ESI) m/z: [M+H]+ calcd for C₁₇H₂₂F₆N₃O₄: 446; found446; RT=2.635 min. ¹H NMR (400 MHz, METHANOL-d₄) δ: 1.24-1.45 (m, 6H),2.29 (s, 2H), 3.31-3.32 (m, 3H), 3.39-3.53 (m, 2H), 5.14-5.25 (m, 1H),6.71 (dd, J=6.72, 1.71 Hz, 1H), 6.81 (s, 1H) and 7.77 (d, J=6.72 Hz, 1H)ppm.

Example 74. Preparation ofN-(5-(6-aminopyridin-3-yl)-1-(isoxazol-3-yloxy)-2-oxopentan-3-yl)cyclopentanecarboxamide(74)

To a mixture of compound 74.8 (5 g, 28.9 mmol, 1 eq) and TEA (11.7 g,115.6 mmol, 16.09 mL, 4 eq) in DMF (100 mL) was added Boc₂O (18.92 g,86.7 mmol, 19.92 mL, 3 eq) in one portion at 25° C. under N₂. Themixture was stirred at 25° C. for 10 hours. The reaction mixture wasdiluted with H₂O (100 mL) then was extracted with EtOAc (100 mL×3). Thecombined organic layers were washed with brine (200 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure. The residuethat remained was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=5:1) to give compound 74.7

(20 g, 53.58 mmol, 46% yield) as a white solid. ¹H NMR (400 MHz,chloroform-d) δ: 1.45 (s, 20H), 7.18 (d, J=8.44 Hz, 1H), 7.81-7.91 (m,1H), 7.81-7.91 (m, 1H) and 8.54 (d, J=2.20 Hz, 1H) ppm.

To a solution of compound 74.6 (3 g, 12.04 mmol, 1 eq) in dioxane (30mL) was added 9-BBN (0.5 M, 53 mL, 2.2 eq) in one portion at 80° C.under N₂. The mixture was stirred at 80° C. for 30 mins, then there wasadded CsF (5.48 g, 36.11 mmol, 1.33 mL, 3 eq), Pd(dppf)Cl₂ (880.65 mg,1.2 mmol, 0.1 eq) and compound 74.7 (5.39 g, 14.44 mmol, 1.2 eq). Thismixture was stirred at 80° C. for 9.5 hours; then the reaction wasdiluted with H₂O (20 mL) and extracted with EtOAc (20 mL×3). Thecombined organic layers were washed with brine (20 mL), dried overNa₂SO₄, filtered and concentrated. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1) to givecompound 74.5 (4 g, 7.36 mmol, 61% yield) as yellow oil. ¹H NMR (400MHz, chloroform-d) δ: 1.44 (s, 18H), 1.90-2.03 (m, 2H), 2.11-2.26 (m,1H), 2.55-2.76 (m, 3H), 3.66-3.80 (m, 4H), 4.37-4.50 (m, 1H), 5.08-5.18(m, 2H), 5.36-5.49 (m, 1H), 7.14 (d, J=7.89 Hz, 1H), 7.31-7.42 (m, 6H),7.55 (br d, J=8.33 Hz, 1H) and 8.30 (s, 1H) ppm.

To a solution of compound 74.5 (3.8 g, 6.99 mmol, 1 eq) in MeOH (100 mL)was added 10% Pd on carbon catalyst (400 mg) under N₂. The suspensionwas degassed under vacuum and purged with H₂ several times. The mixturewas stirred under H₂ atmosphere (50 psi) at 25° C. for 0.5 hour. Thereaction mixture was purged with N₂ gas, filtered through a Celite® padand was concentrated under reduced pressure to give a residue. Theresidue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=10:1 to 1:1) to give compound 74.4 (2 g, 4.88 mmol,70% yield) as a yellow solid.

To a mixture of cyclopentanecarboxylic acid (111.5 mg, 976.86 μmol,106.19 μL, 1 eq) and EDCI (206 mg, 1.07 mmol, 1.1 eq) in DMF (25 mL) wasadded HOBt (145.19 mg, 1.07 mmol, 1.1 eq) in one portion at 0° C. underN₂. The mixture was stirred at 0° C. for 1 hour. After this time, to themixture was added dropwise a solution of compound 74.4 (400 mg, 976.86μmol, 1 eq) in DMF (5 mL), then there was added dropwise DIPEA (378.75mg, 2.93 mmol, 510.44 μL, 3 eq). This was stirred at 0° C. for 1 hour;then the reaction mixture was diluted with H₂O (10 mL) and extractedwith EtOAc (10 mL×3). The combined organic layers were washed with brine(15 mL), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=3:1) to givecompound 74.3 (2 g, 3.96 mmol, 81% yield) as a white solid. ¹H NMR (400MHz, chloroform-d) δ: 1.44 (s, 15H), 1.59 (br d, J=6.11 Hz, 2H), 1.67-2(m, 8H), 2.12-2.28 (m, 1H), 2.50-2.77 (m, 3H), 3.66-3.82 (m, 3H),4.63-4.75 (m, 1H), 6-6.12 (m, 1H), 7.15 (d, J=8.07 Hz, 1H), 7.51-7.61(m, 1H) and 8.29 (s, 1H) ppm.

To a solution of DIPA (2.20 g, 21.76 mmol, 3.07 mL, 5.5 eq) in THF (10mL) was added n-BuLi (2.5 M, 8.70 mL, 5.5 eq) at 0° C., the mixture wasstirred at 0° C. for 30 mins. To this mixture was added a solution ofchloroiodomethane (3.84 g, 21.76 mmol, 1.58 mL, 5.5 eq) and compound74.3 (2 g, 3.96 mmol, 1 eq) in THF (10 mL) at −78° C. The mixture wasstirred at −78° C. for 30 mins. The reaction mixture was quenched byaddition of NH₄Cl (20 mL) and then extracted with EtOAc 60 mL (20 mL×3).The combined organic layers were washed with saturated aqueous Na₂SO₃(30 mL), and brine (50 mL), dried over Na₂SO₄, filtered and concentratedunder reduced pressure to give a residue. The residue was purified bycolumn chromatography (SiO₂, Petroleum ether/Ethyl acetate=2:1) to givecompound 74.2 (500 mg, 954.11 μmol, 24% yield) as yellow oil. LCMS (ESI)m/z: [M+H]+ calcd for C₂₆H₃₉N₃O₆Cl: 525; found 525; RT=1.33 min.

To a mixture of compound 74.2 (500 mg, 954.11 μmol, 1 eq) andisoxazol-3-ol (81.16 mg, 954.11 μmol, 1 eq) in DMF (4 mL) was added DIEA(369.93 mg, 2.86 mmol, 498.56 μL, 3 eq) in one portion at 25° C. underN₂. The mixture was stirred at 25° C. for 10 hours. The reaction waspurified by prep-HPLC (TFA condition) to give compound 74.1 (100 mg,211.63 μmol, 22% yield) as yellow oil. ¹H NMR (400 MHz, chloroform-d) δ:1.56 (s, 10H), 1.62 (br s, 2H), 1.70-1.83 (m, 4H), 1.85-1.97 (m, 3H),2.24-2.36 (m, 1H), 2.56-2.66 (m, 1H), 2.70 (br t, J=7.95 Hz, 2H),4.82-4.89 (m, 1H), 4.92-5.07 (m, 2H), 6.08 (s, 1H), 6.22 (br d, J=7.70Hz, 1H), 7.92 (br d, J=9.29 Hz, 1H), 7.99 (s, 1H), 8.17 (s, 1H) and 8.35(d, J=8.68 Hz, 1H) ppm.

To a solution of compound 74.1 (100 mg, 211.63 μmol, 1 eq) in DCM (5 mL)was added TFA (211.63 μmol, 15.67 μL, 1 eq) in one portion at 25° C.under N₂. The mixture was stirred at 25° C. for 1 hour. The reactionmixture was concentrated under reduced pressure to give a residue. Theresidue was purified by semi-preparative scale HPLC (TFA condition) togive 74 (40 mg, 107.41 μmol, 51% yield) as colorless oil. LCMS (ESI)m/z: [M+H]+ calcd for C₁₉H₂₅N₄O₄: 373; found 373; RT=2.046 min. ¹H NMR(400 MHz, METHANOL-d4) δ: 1.56-1.67 (m, 2H), 1.69-1.81 (m, 4H),1.84-1.97 (m, 3H), 2.14-2.25 (m, 1H), 2.53-2.81 (m, 3H), 4.49 (dd,J=9.17, 4.77 Hz, 1H), 4.95-5.11 (m, 2H), 6.15 (d, J=1.83 Hz, 1H), 6.99(d, J=9.17 Hz, 1H), 7.65 (d, J=1.34 Hz, 1H), 7.88 (dd, J=9.17, 2.20 Hz,1H) and 8.37 (d, J=1.83 Hz, 1H) ppm.

Example 75. Inhibition of Arginine Gingipain by theAminopyridinecyanamide Compounds of the Invention

The capacities of compounds of the present invention to inhibit theactivity of RgpB were measured in a fluorogenic assay similar to thosedescribed in Barret Biochemical Journal. 1980, 187(3), 909. The specificassay conditions were as follows. Buffer: pH=7.5, 100 mM Tris-HCl, 75 mMNaCl, 2.5 mM CaCl₂, 10 mM cysteine, 1% DMSO after all additions.Protein: 0.02 nM RgpB, isolated from culture of Porphyromonasgingivalis, as described in Pike et al. J. Biol. Chem. 1994, 269(1),406, and Potempa and Nguyen. Current Protocols in Protein Scienc. 2007,21.20.1-21.20.27. Fluorogenic substrate: 10 μM Boc-Phe-Ser-Arg-MCA.Time=90 minutes. Temperature=37° C. Each compound: 10 concentrations,starting at either 100 μM or 100 nM, with lower concentrations generatedby serial 3-fold dilutions. By testing a range of concentrations foreach compound, the concentration required to inhibit the activity ofRgpB by 50% (the “IC₅₀”) was determined. RgpB inhibitory activity issummarized in the following table.

No. Activity  1 +  2 ++++  3 ++++  4 ++  5 ++  6 ++  7 +  8 +  9 ++++10 + 11 + 12 ++++ 13 ++++ 14 ++++ 15 ++++ 16 ++ 17 +++ 18 ++ 19 +++++ 20+++ 21 ++++ 22 ++++ 23 ++ 24 ++ 25 ++++ 26 ++++ 27 + 28 +++ 29 ++ 30 ++31 + 32 ++++ 33 +++ 34 +++ 35 +++ 36 +++ 37 ++++ 38 ++ 39 ++ 40 ++ 41 ++42 ++ 43 +++++ 44 ++++ 45 +++++ 46 +++ 47 +++ 48 ++++ 49 +++++ 50a +++++50b +++++ 51a ++ 51b ++ 52a >1 μM^(a) 52b >1 μM^(a) 53 +++++ 54 +++++ 55+++++ 56 +++++ 57 +++++ 58 ++++ 59 ++++ 60 +++++ 61 +++++ 62 ++++ 63 +++64 ++++ 65 +++++ 66 +++++ 67 +++++ 68 +++++ 69 +++++ 70 ++++ 71 ++++72 >1 μM^(a) 73 >1 μM^(a) 74 +++++ +++++: RgpB IC₅₀ < 100 nm ++++: 100nM < RgpB IC₅₀ < 1 μM +++: 1 μM < RgpB IC₅₀ < 10 μM ++: 10 μM < RgpBIC₅₀ < 100 μM +: 100 μM < RgpB IC₅₀ ^(a)compound not tested above 1 μM

Although the foregoing has been described in some detail by way ofillustration and example for purposes of clarity and understanding, oneof skill in the art will appreciate that certain changes andmodifications can be practiced within the scope of the appended claims.In addition, each reference provided herein is incorporated by referencein its entirety to the same extent as if each reference was individuallyincorporated by reference.

What is claimed is:
 1. A compound according to Formula III:

or a pharmaceutically acceptable salt thereof, wherein: R¹¹ and R^(12a)are taken together to form 4- to 10-membered heterocyclyl which isoptionally substituted with one or more R¹⁷; R^(12b) is H; R^(13a) andR^(13b) are independently selected from the group consisting of H, —OH,and C₁₋₆ alkyl; R¹⁴ is H, or R¹⁴ and R^(13a) are taken together to forma 5- to 8-membered ring, which is optionally substituted with one ormore R¹⁸; R^(15a) and R^(15b) are H; R¹⁶ is independently selected fromthe group consisting of C₁₋₆ alkyl and halogen; Y is selected from thegroup consisting of O, S, C(R^(19a))₂, and NR^(19b); each R¹⁸ isindependently selected from the group consisting of C₁₋₆ alkyl andhalogen; each R^(19a) is selected from the group consisting of H andC₁₋₆ alkyl, or one R^(19a) and R^(13b) are taken together to form adouble bond; R^(19b) is selected from the group consisting of H and C₁₋₆alkyl; subscript m is 0 or 1; and subscript q is 0 or
 1. 2. The compoundof claim 1, or a pharmaceutically acceptable salt thereof, wherein Y isC(R^(19a))₂, and wherein one R^(19a) and R^(13b) are taken together toform a double bond.
 3. The compound of claim 2, or a pharmaceuticallyacceptable salt thereof, wherein R¹⁴ and R^(13a) are taken together toform a 5- to 8-membered ring.
 4. The compound of claim 3, or apharmaceutically acceptable salt thereof, wherein R^(12a) and R¹¹ aretaken together to form pyrrolidin-1,2-diyl.
 5. The compound of claim 4,or a pharmaceutically acceptable salt thereof, wherein subscript q is 0.6. A pharmaceutical composition comprising a compound of claim 1, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient.
 7. A method of treating a disease or conditionassociated with P. gingivalis infection, the method comprisingadministering to a subject in need thereof an effective amount of claim1, or a pharmaceutically acceptable salt thereof.
 8. The method of claim7, wherein the disease or condition is selected from the groupconsisting of a brain disorder, periodontal disease, diabetes, acardiovascular disease, arthritis, rheumatoid arthritis, osteoarthritis,infectious arthritis, psoriatic arthritis, elevated risk of pretermbirth, pneumonia, cancer, a kidney disease, a liver disease, a retinaldisorder, and glaucoma.
 9. The method of claim 8, wherein the disease orcondition is a brain disorder.
 10. The method of claim 9, wherein thebrain disorder is Alzheimer's disease.
 11. The method of claim 10,further comprising administering to the subject one or more activeagents selected from the group consisting of a cholinesterase inhibitor,a serotonin modulator, an NMDA modulator, an Aβ targeted therapy, anApoE targeted therapy, a microglia targeted therapy, a blood brainbarrier targeted therapy, a tau targeted therapy, a complement targetedtherapy, and an anti-inflammatory.
 12. The method of claim 8, whereinthe disease or condition is periodontal disease.
 13. The method of claim7, wherein the compound is administered to the subject for at least oneyear.
 14. The method of claim 13, wherein the compound is administeredto the subject for at least 10 years.
 15. The method of claim 7, whereinthe subject is a human, a canine, or a feline.